• Journal of Embryo Transfer
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• v.33 no.4
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• pp.211-220
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• 2018

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to $0.1{\mu}M$ SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and $10{\mu}M$ during the first 22 h of IVM to determine a suitable concentration, $0.1{\mu}M$ SNAP (54.2%) exhibited a higher blastocyst formation than 0 and $10{\mu}M$ SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.26-31
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• 2004

This work was undertaken in order to study the developmental competence of nuclear transfer cat embryo with fetal fibroblast and adult skin fibroblast as donor nuclei. Oocytes wererecovered by mincing the ovaries in Hepes-buffered TCM199 and selected the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark. Homogenous ooplasm were cultured for maturation in TCM199 + 10% fetal bovine serum (FBS) for 12 hours and used as a source of recipient cytoplast for exogenous somatic nuclei. In Experiment 1, we evaluated the effect donor cell types on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate was not different between fetal fibroblast and adult skin cell (71.2 vs. 66.8; 71.0 vs. 57.6; 4.0 vs. 6.1 %, P<0.05). In Experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of seven recipient queens was delivered naturally 2healthy cloned cats and 1 stillborn from fetal fibroblast cell of male origin after 65 days embryo transfer. One of three recipient queens was delivered naturally 1 healthy cloned cat from adult skin cell of female after 65 days embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.2
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• pp.45-50
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• 2015

Objective: Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. Methods: The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). Results: The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. Conclusion: We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.81-87
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• 2004

This study was designed to determine whether repeated superovulation is beneficial for recovery and quality of oocytes in Korean native goats. Seventy-six mature goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen impregnated CIDR for 10 days and then the goats were divided into two groups. One group of the goats received a single intramuscular injection of 1,000 IU PMSG on Day 8 of CIDR insertion. The other group of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF2{\alpha}on$ Day 8 and 400 IU hCG in the afternoon on Day 10. For oocyte recovery, donor goats were fasted 24 h before operation. Anesthesia was induced by intravenous injection of 2% xylazine(0.2 mg/kg body weight) and ketamin(11 mg/kg body weight). In vivo oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 hours after hCG injection through mid-ventral incision. The mean number of CL and oocytes recovered and recovery rate of oocytes by oviduct flushing were greater(P<0.05) in the first treatment than those in the second treatment. Contrary to our assumption, PMSG treatment significantly (P<0.05) increased the number of CL formed and recovery rate of oocytes compared to FSH. However, the same effect was not observed in recovery of follicular oocytes. There was no significant difference in oocyte quality between FSH and PMSG or first and second treatments. The present results indicate that repeated superovulation and repeated use of donor animals may be inefficient for obtaining oocytes in good qualities.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.7
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.227-232
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• 2020

Objective: The aim of this study was to compare in vitro fertilization outcomes between fresh day 3 or day 4 embryo transfer cycles with dual progesterone (P) administration (intramuscular and vaginal) and cycles with single intramuscular P administration for luteal support. Methods: We selected 124 cycles from 100 women (under age 40 years) who underwent oocyte pick-up (number of trials ≤ 3, 4-14 oocytes obtained) and transfer of two or three day 3 or day 4 embryos at two infertility centers from January 2014 to June 2019. Dual P (intramuscular P [50 mg] daily+vaginal P) was used in 52 cycles and a single intramuscular administration of P (50 mg daily) was used in 72 cycles. Results: Women's age, infertility factors, number of oocytes retrieved, number of transferred embryos, and mean embryo score were similar between the dual P group and the single P group. Although the number of trial cycles was significantly higher (1.9 vs. 1.5), and the mean endometrial thickness on the trigger day (10.0 mm vs. 11.0 mm) was significantly lower in the dual P group, the implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and miscarriage rate for both day 3 and day 4 transfers were similar between the two groups. Conclusion: In fresh day 3 or day 4 embryo transfer cycles, dual P administration did not demonstrate any clinical advantages. Intramuscular P alone appears to be sufficient for luteal support.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.8-13
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• 2015

Objective: Great advances have been made in the field of assisted reproductive technology (ART) since the first in vitro fertilization (IVF) baby was born in Korea. This study was designed to report on the current status of ART therapy in South Korea between January 1 and December 31 of 2010. Methods: A revised survey, originally developed by the International Committee Monitoring Assisted Reproductive Technologies, was sent to all available ART centers via email in 2013. Fresh embryo transfer (FET) cases were categorized into standard IVF or intracytoplasmic sperm injections. These cases, the thawing embryo transfer (TET) cases, and other related procedures were surveyed. Results: Data from 30,785 ART procedures were provided by 78 clinics. Of the 28,200 cycles in which oocytes were retrieved, 92.2% of these cycles were completely transferred. In addition, 8,075 cycles were confirmed to be clinical pregnancies in the FET cycles, which represent a pregnancy rate of 28.6% per oocyte pick-up and 31.1% per embryo transfer. The most common number of embryos transferred in the FET was three embryos (37.3%) followed by two embryos (36.3%) and one embryo (14.0%). Of the 6,648 TET cycles transferred, 2,356 clinical pregnancies were confirmed by ultrasonography. The most common number of embryos in the TET group was two embryos (43.4%) followed by three embryos (25.4%) and one embryo (18.9%). Conclusion: The clinical pregnancy rate per transfer in the FET cycles was similar in 2009 and 2010. Among the FET cycles where one or two embryos were transferred, the clinical pregnancy rate per transfer slightly increased from 2009 (28.7%) to 2010 (32.9%).

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.235-243
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• 2007

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional $10{\sim}14$ h. Control oocytes were denuded after $40{\sim}44$ h of IVM. The size of the perivitelline space was larger at 40 h of IVM ($11.7{\sim}11.8{\mu}m$) than at 30 h ($8.9{\mu}m;$ p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ ($19.4{\mu}m$) and D- oocytes ($18.9{\mu}m$) than in control oocytes ($25.5{\mu}m;$ p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

• Journal of Animal Science and Technology
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• v.66 no.5
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• pp.920-935
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• 2024

Embryo transfer plays a crucial role in enhancing the breeding value of livestock; it has been applied in Hanwoo cattle, which is a popular breed for beef production in Korea. Both in vivo-derived (IVD) and in vitro-produced (IVP) embryos are used for this purpose; however, IVP embryos have been preferred recently owing to advancements in ovum pick-up (OPU) technology and genomic selection. Despite technological advancements, comprehensive data on large-scale OPU/IVEP/embryo transfer in Hanwoo cows are lacking. In this study, 16 elite Hanwoo donor cows were selected on the basis of specific criteria. Oocytes were retrieved from 241 cows using OPU. The collected cumulus-oocyte complexes (COCs) were matured, fertilized, and cultured in vitro to produce transferable embryos. Embryos were classified according to their developmental stage and then transferred to 675 recipient cows. A total of 3,317 COCs were collected, with an average of 13.76 COCs per cow. The number of transferable embryos produced per cow was 3.7. Hanwoo OPU-derived IVP embryos exhibited a higher production yield than the global average, indicating a stable IVEP environment. Both fresh and frozen IVP embryos yielded similar conception rates; hence, the use of vitrified-thawed embryos in transfer plans feasible. However, frozen-thawed embryos at Stage 7 had a lower conception rate than those at earlier stages. There was no significant difference between the conception rates of sexually mature heifers and postpartum cows used as recipients. The male-to-female offspring ratio increased as the developmental stage progressed. Seasonal effects on conception rates were not observed; however, higher abortion rates and a higher proportion of male offspring were observed during winter. This study provides valuable data for the Korean embryo transfer industry, enabling more strategic growth of the domestic Hanwoo embryo industry.