• Journal of Embryo Transfer
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• v.20 no.1
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• pp.25-33
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• 2005

This study is a part of research that development of effective genetic resources preservation system using the in vitro spermatogenesis, in vitro insemination and culture system. We aimed for establishment of in vitro culture system with in vitro activated porcine oocytes. The porcine oocytes were matured for 48 hours in $TCM199+10\%$ FCS and activated with $7\%$ ethanol. The activated oocytes were cultured for 7 days in $TCM199+10\%$ FCS or $NCSU23+0.4\%$ BSA medium. The activated oocytes were not developed to the blastocyst stage in $TCM199+10\%$ FCS medium. However in $NCSU23+0.4\%$ medium, those were developed to blastocyst with $3\%$ of treated oocytes. We extended maturation duration of porcine follicular oocytes fur 48, 52, 56, 60, 64, 68, and 72 hours and activated with $7\%$ ethanol and cultured using $NCSU23+0.4\%$ BSA medium. The six percents of activated oocytes were developed to blastocyst in 48 hours and $10\%$ in 52 hours with comparatively low rates suggested to be not fully activated by regenerated MPF. Maturation durations from 56 hours to 68 hours supported to develop upto $11.9\~18.3\%$ of blastocysts. However the developmental rate was declined to $7.2\%$ at 72 hours of maturation duration because of cytoplasmic deterioration. The assumed time window for activation will be $56\~68$ hours of maturation duration. When the matured oocytes were activated with electric pulse of 1, 1.2, 1.4, 1.6, 1.8 and 2.0kV/cm for $80{\mu}s$, although appling the electric current once was not enough for activation, appling twice with 1.6kV/cm for $80{\mu}s$ was shown the highest developmental rate with $11.3\%$. When those were compared with activating methods, $15.7%$ of blastocyst rate was obtained in the $7\%$ ethanol. That was higher than those in electric pulse with $9.5\%$ and calcium ionophore method with $5.8\%$. In this experimental condition, the $7\%$ ethanol treatment was the most effective method for activating porcine oocytes.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.345-351
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• 2006

The objective of this study was to investigate the effects of preservation period of porcine liquid semen on bacterial contamination and in vitro production of embryo. Extended liquid semen was prepared by three mixture of boar's ejaculates from each farm without antibiotics, and were kept in $17^{\circ}C$ semen preservation incubator until use. Sperm motility was significantly (p<0.05) decreased as semen preservation time goes by (78.7$\pm$2.4% for 1 day vs. 71.1$\pm$2.4 and 64.8$\pm$2.4% for 3 and 5 days of presentation, respectively). Quantitative of bacteria in semen was significantly (p<0.05) higher in 5 days ($57.8\pm105.2\times10^4$ Cfu) compared to 0 and 3 days ($32.1\pm76.8\times10^4$ and $26.9\pm46.6\times10^4$ Cfu, respectively) of preservation. In terms of development of in vitro fertilization of porcine embryos inseminated by preserved semen, the rate of normal fertilization (2PN) was significantly (p<0.05) decreased in 5 days (56.0$\pm$2.6%) compared to 1 and 3 days (66.0$\pm$2.7 and 64.0$\pm$2.7%, respectively) of preservation. Cleavage rate was also significantly (p<0.05) affected by preservation period (75.0$\pm$4% for 1 day, 70.0$\pm$0.3 and 71.0$\pm$0.3% for 3 and 5 days, respectively). The in vitro developmental rate of blastocyst stage embryo was significantly (p<0.05) affected by semen preservation period (15.0$\pm$1.0% for 1 day vs. 11.0$\pm$0.9 and 8.0$\pm$0.9% for 3 and 5 days, respectively). It is concluded that more than 3 days of liquid semen preservation without antibiotics increased the quantity of bacteria resulted in detrimental effect on sperm motility and decreased both normal insemination rate and the developmental rate of blastocyst stage embryo.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.115-124
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• 2003

The objective of this study was to compare the effect of four different estrus induction methods in anestrus cows on the estrus induction and pregnancy following artificial insemination (AI). Sixty-five cows (3∼4 years old) were selected and divided into four different estrus induction treatment groups. Group 1, 12 cows were treated by Ovsynch program combined with GnRH and PGF$_2$a. Group 2, 12 cows were treated by "Tow plus Two" program with GnRH and PGF$_2$a. Group 3, 20 cows were treated by "Tow plus Two" program following intravaginal progesterone implantation (CIDR). Twenty one cows in Group 4 were treated by "Tow plus Two" program following follicular rupture and intravaginal progesterone implantation. Cows were then observed estrus induction and inseminated artificially at 12 h and 24 h after standing estrus. The rates of estrus induction in Group 4 (18/21, 86%) was significantly (P<0.05) higher than those in groups 1, 2 and 3 (8/12, 67%; 9/12, 75%; 14/20, 70%). In the mean time of onset of estrus after final administration of GnRH in different hormone-treated cows, the cows in Group 3 (24.2$\pm$2.2) and Group 4 (23.4$\pm$2.0) were significantly (P<0.05) shorter than that in Group 1 (28.5$\pm$4.6) and Group 2 (26.4$\pm$3.3). The rates of pregnancy diagnosed on Day 28 were significantly different between treatment groups. Significantly (P<0.05) higher rate of pregnancy was observed in Group 4 (17/20, 85.0%) than those in Groups 1, 2 and 3 (7/11, 63.6%; 8/12, 66.7%; 15/20, 75.0%, respectetively). The rate of abortion diagnosed on 49 days of gestation was significantly (P<0/05) lower in Group 4 (1/17, 5.9%) than those in Groups 1, 2 and 3 (2/7, 28.7%; 2/8, 25% and 3/15, 20%, respectively). In conclusion, combined treatments with GnRH and PGF$_2$a following follicular rupture and progesterone implant in anestrus cows was considered to be most effective in estrus induction and maintenance of pregnancy. Further studies are needed to verify the functional mechanisms of residual follicles in anestrus ovaries on retarding the response of hormonal treatments.sponse of hormonal treatments.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.169-176
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• 2005

Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.