• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.313-321
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• 1999

The purpose of these study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of bovine IVP blastocysts, to compare the post-thaw survival rates of bovine blastocysts frozen in GMP with those frozen in OPS that have been previously investigated, and to improve the hatching rate following vitrification with GMP method. The GMP vessel permits higher freezing and warming rate than the OPS due to the higher heat conductivity of the glass and lower mass of the solution that contains the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or 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 each 5 min, respectively, and then cultured in TCM 199 supplemented with 10% FCS for 24 h. The rate of blastocyst re-expanding did not significantly different for OPS (75.9%) and GMP (80.0%) methods (P＞0.05). The hatching rates in OPS (34.1%) and GMP (37.5%) methods were significantly lower than that in control group (54.3%) (P＞0.05). In addition, the rate of blastocyst re-expanding was significantly lower if blastocysts were vitrified in the wide portion of the micropipette rather than the narrow portion of the micropipette (83.3 vs 56.7%) (P＞0.05), even though three blastocysts were loaded per vessel. The hatching rate in 0.05% pronase solution treatment for 30, 60 and 90 see (45.9, 54.7 and 57.5%) were significantly higher than that in control (35.0%), even though there was not significantly different between 30 see and control. These results indicate that both vitrification vessels can provide high survival rates of bovine IVP blastocysts. However, the GMP vessel has the advantage over the OPS, in that the former does not need a cap to protect the vessel from floating after immersion in L$N_2$. The location of the embryos (narrow or wide portion of immersion) were considered to be limiting factors to the viability of bovine IVP embryos. The exposing in 0.05% pronase solution for 60 or 90 see can increase hatching rates of post-thaw bovine IVP blastocysts.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.129-136
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• 2002

The purpose of this study was to investigate the comparison of viability of bovine blastocysts following glass micropipette (GMP) vitrification and the possibility of production of Korean Native Cow ("Hanwoo,"Bos taurus coreanae) following embryo transfer into Mongolia cows (Bos taurus mongolian). The embryos of Korean Native Cow were produced by IVMFC or superovulation methods in Korea, cryopreserved by GMP vitrification, and subsequently trans-ported to Mongolia. The recipient cows were synchronized using a CIDR plus and prostaglandin $F_2\alpha$($PGF_2\alpha$) treatment. To produce in vivo embryos, seven cows were superovulated using FSH and PGF$_2$／sub $\alpha$／ treatment. A total of 64 blastocysts ( $9.1\pm2.94$ per cow) were collected. In vitro embryos were produced using a defined culture system which cleaved in 80.1％ ova (174／217), and developed to blastocyst stage embryos of 40.8％ (71／174). The post-thaw survival rate of in vivo blastocysts (93.7％; 45／48) was significantly higher than that of in vitro blastocysts (82.5％; 52／63, P＜0.05). Embryo transfer was carried out using 8 Mongolian recipient cows and 2 post-thaw blastocysts per recipient. Five of 8 recipients were found pregnant at Day 60 but one abortion occurred by Day 240. Two of offspring were produced from the Mongolian cows at 275 days after embryo transfer. These results indicated that a GMP vitrification method could be used as a cryopreservation technique for in vivo or in vitro bovine blastocysts and produced effectively a Korean Native Cow following embryo transfer into a Mongolian recipient cow.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.83-83
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• 2002

The purpose of this study was to investigate the warming temperature and exposed time on the post-thaw survival rate and viability of bovine blastocyst cryopreserved by GMP vitrification. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into the GMP straws and immersed into LN$_2$within 20 to 25 sec. The warming rate was increased 2 times of warming temperature for improvement of post-thaw survival rates. The frozen embryos were warmed either at 35 or 70$^{\circ}C$ for 1 or 2 sec and then diluted in sucrose solution. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM: TCM199 supplemented with 10% FCS) and TCM-199 for each 5 min, respectively, and then cultured in TCM199 for 24 h. The rate of re-expanded blastocyst was significantly different fer 35 and 70$^{\circ}C$ warming temporature (76.4 vs. 89.3%; P<0.05). The rate of re-expanded blastocyst at 70$^{\circ}C$ for 1 sec was significantly higher than that for 2 sec (91.1 vs. 70.9%; P<0.05). The number of nuclei counted were significantly different among control, 35 and 70$^{\circ}C$ (121${\pm}$8.5 vs. 104${\pm}$11.7 vs. 114${\pm}$10.3; P<0.05). These results indicated that the increasing of warming rate can provide high survival rates of bovine IVP blastocysts. Especially, the best viability of post-thaw blastocyst could be thaw at 70$^{\circ}C$ for 1 sec. The warming temperature and exposed time far warming was considered to be limiting factors to the viability of bovine IVP embryos. he purpose of this study was to investigate the warming temperature and expose.