• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.191-198
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• 2001

The purpose of this study was to investigate the effects of embryo sources such as in vivo vs. in vitro produced blastocyst, and culture systems on the membrane permeability and viability of bovine blastocyst following GMP vitrification. To produce in vivo embryos, six cows were superovulated by administration of follicle stimulation hormone (FSH) and prostaglandin $F_{2{\alpha}}$(PG $F_{2{\alpha}}$). in vitro embryos were produced by two different culture systems, oviduct co-culture (OCS) and defined culture system (HECM-6; DCS). Ovaries were picked up at a local slaughterhouse and transported to laboratory in 3$0^{\circ}C$ saline within 2 h. Ovaries were washed with same saline three times and then placed in saline on warm plate adjusted at 3$0^{\circ}C$ during aspiration. The blastocysts produced were assigned for membrane permeability and viability following GMP vitrification. The membrane permeability of blastocysts was checked in 0.5 M sucrose solution on warm plate at 35$^{\circ}C$ for 0, 2, 5 and 7 min, respectively. Then the diameters (width and length) of embryo cytoplasms were measured by a eyepiece meter, and they were converted to their volume by 4/3 $\pi$ $r^3$. The blastocysts were cryopreserved by GMP vitrification method, where they were sequentially placed into vitrification solution before being loaded into GMP vessels and immersed into L$N_2$ within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in HM and TCM-199 for 5 min each, and then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The volume change of in vivo blastocyst at 0, 2, 5 and 7 min (100, 37.1, 34.3 and 31.6%) was significantly more shrunk than those of in vitro blastocysts derived from OCS (100, 59.8, 48.9, 47.9%) and DCS (100, 57.2, 47.3 and 46.9%) (P＜0.05). The viability of post-thaw blastocyst derived from in vivo (93.6%) was also significantly different from those in OCS and DCS (81.9 and 83.6%; P＜0.05). In the present culture system, the morphology of embryos produced in vitro was similar to that of in vivo embryos, but the quality in membrane permeability and post-thaw viability showed a big difference from their sources as in vivo or in vitro derived from OCS and DCS. The results indicated that the quality of in vivo embryos in membrane permeability and post-thaw viability was better than those of in vitro embryos derived from OCS or DCS.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.85-92
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• 1999

This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

• Journal of The Korean Astronomical Society
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• v.12 no.1
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• pp.41-70
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• 1979

From $B\ddot{o}hm$-Vitense's atmospheric model calculations, the relations, [$T_e$, (B-V)] and [B.C, (B-V)] with respect to heavy element abundance were obtained. Using these relations and evolutionary model calculations of Rood, and Sweigart and Gross, analytic expressions for some physical parameters relating to the C-M diagrams of globular clusters were derived, and they were applied to 21 globular clusters with observed transition periods of RR Lyrae variables. More than 20 different parameters were examined for each globular cluster. The derived ranges of some basic parameters are as follows; $Y=0.21{\sim}0.33,\;Z=1.5{\times}10^{-4}{\sim}4.5{\times}10^{-3},\;age,\;t=9.5{\sim}19{\times}10^9$ years, mass for red giants, $m_{RG}=0.74m_{\odot}{\sim}0.91m_{\odot}$, mass for RR Lyrae stars, $m_{RR}=0.59m_{\odot}{\sim}0.75m_{\odot}$, the visual magnitude difference between the turnoff point and the horizontal branch (HB), ${\Delta}V_{to}=3.1{\sim}3.4(<{\Delta}V_{to}>=3.32)$, the color of the blue edge of RR Lyrae gap, $(B-V)_{BE}=0.17{\sim}0.21=(<(B-V)_{BE}>=0.18),\;[\frac{m}{L}]_{RR}=-1.7{\sim}-1.9$, mass difference of $m_{RR}$ relative to $m_{RG},(m_{RG}-m_{RR})/m_{RG}=0.0{\sim}0.39$. It was found that the ranges of derived parameters agree reasonably well with the observed ones and those estimated by others. Some important results obtained herein can be summarized as follows; (i) There are considerable variations in the initial helium abundance and in age of globular clusters. (ii) The radial gradient of heavy element abundance does exist for globular clusters as shown by Janes for field stars and open clusters. (iii) The helium abundance seems to have been increased with age by massive star evolution after a considerable amount (Y>0.2) of helium had been attained by the Big-Bang nucleosynthesis, but there is not seen a radial gradient of helium abundance. (iv) A considerable amount of heavy elements ($Z{\sim}10{-3}$) might have been formed in the inner halo ($r_{GC}$<10 kpc) from the earliest galactic co1lapse, and then the heavy element abundance has been slowly enriched towards the galactic center and disk, establishing the radial gradient of heavy element abundance. (v) The final galactic disk formation might have taken much longer by about a half of the galactic age than the halo formation, supporting a slow, inhomogeneous co1lapse model of Larson. (vi) Of the three principal parameters controlling the morphology of C-M diagrams, it was found that the first parameter is heavy clement abundance, the second age and the third helium abundance. (vii) The globular clusters can be divided into three different groups, AI, BI and CII according to Z, Y an d age as well as Dickens' HB types. BI group clusters of HB types 4 and 5 like M 3 and NGC 7006 are the oldest and have the lowest helium abundance of the three groups. And also they appear in the inner halo. On the other hand, the youngest AI clusters have the highest Z and Y, and appear in the innermost halo region and in the disk. (viii) From the result of the clean separations of the clusters into three groups, a three dimensional classification with three parameters, Z, Y and age is prsented. (ix) The anomalous C-M diagrams can be expalined in terms of the three principal parameters. That is, the anomaly of NGC 362 and NGC 7006 is accounted for by the smaller age of the order of $1{\sim}2{\times}10^9$ years rather than by the helium abundance difference, compared with M 3. (x) The difference in two Oosterhoff types I and II can be explained in terms of the mean mass difference of RR Lyrae variables rather than in terms of the helium abundance difference as suggested by Stobie. The mean mass of the variables in Oosterhoff type I clusters is smaller by $0.074m_{\odot}$ which is exactly consistent with Rood's estimate. Since it was found that the mean mass of RR Lyrae stars increases with decreasing Z, the two Oosterhoff types can be explained substantially by the metal abundance difference; the type II has Z<$3.4{\times}10^{-4}$, and the type I has higher Z than the type II.