• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.91-91
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• 2002

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to develop a treatment protocol for estrus induction. Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifane (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. After being treated, the dogs were evaluated for the rates of estrus induction and time interval lapses from treatment to beginning of the pro-estrus. The estrus induction rates were significantly (P<0.05) higher in both group 2 (9/15, 73.3%) and group 3 (16/20, 80.0%) than that of group 1 (9/15, 60.0%), but did not differ in the groups 2 and 3. No differences were observed in the time interval lapses from treatment to beginning of the pro-estrus in group 2 (7.7 ${\pm}$ 1.2 days) and group 3 (6.9 ${\pm}$ 2.0 days), but significantly (P<0.05) shorter than that of group 1 (9.5 ${\pm}$ 2.1 days). In conclusion, the estrus induction rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.9-17
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• 1996

This study was conducted to investigate freezability of in vitro and in vitro matured rabbit oocytes, possibility of NT using frozen-thawed unfertilized oocytes, and NT efficiency by zona-slit micromanipulation. After freezing of in vitro matured oocytes, 33 to 49% of oocytes appeared normal morphology and 1.0M DMSO and 1.5M glycerol showed slightly high survival rate, but there was no difference in survival between two cryoprotectants. Freezability of in vitro matured oocytes was low in 1.5M glycerol and more sensitive to freezing. Efficiency of enucleation and fusion rate in method B was higher than that in method A and no difference in this efficiency was between 3 groups of oocytes in method B. Cleavage rate and developmental capacity to M＋B stage of fused embryos derived from frozen oocytes was greatly lower than that from fresh oocytes, respectively(39.1% : 79.5% ; 3.1% : 19.3%) and there was no difference in cleavage rate between DC voltages in two group oocytes. Additional incubation in cytochalasin B after electrical stimulation did not affect embryo development. In conclusion, it is suggested that enucleation and nucelar transfer by slitting of zona is more effective method in rabbit and that further study on optimum freezing conditions for in vitro matured oocytes is necessary to use as recipient oocytes.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.301-310
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• 2009

Objective: To compare the embryonic development and pregnancy results using sperms retrieved from fresh and frozen-thawed testicular tissue in patients with obstructive (OA) and non-obstructive azoospermia (NOA). Methods: A total two hundred twenty-two cycles of TESE-ICSI were performed in OA and NOA. Sperms were retrieved from fresh and frozen-thawed testicular tissue. ICSI was performed patient's own sperm. Fertilization was assessed 16~18 hrs after ICSI. Embryo development and pregnancy rates were analysed. Results: The fertilization rates were significantly different between OA and NOA patients (75.2% vs. 56.7%, p<0.05), however, embryo development did not differ between the groups (96.9% vs. 98.0%). Likewise, OA and NOA groups had no differences in their clinical pregnancy and delivery rates, 33.9% vs. 36.0% and 28.1% vs. 28.0%, respectively. With regard to sperm retrieved from fresh testicular tissue, fertilization rates were significantly different between the OA and NOA groups (76.4% vs. 52.9%, p<0.05); however, embryo development, clinical pregnancy and delivery rates were not different. For sperm retrieved from thawed testicular tissue, the fertilization rates were significantly different between the two groups (74.7% OA group vs. 65.6% NOA group, p<0.05); however, embryo development, clinical pregnancy and delivery rates were not different. Conclusions: Embryo development and clinical pregnancy did not differ in patients with obstructive and non-obstructive azoospermia, whether sperm retrieved from fresh and thawed testicular tissue were used, although the fertilization rates were different. Therefore, ICSI with sperm retrieved from fresh and thawed testicular tissue could achieve relevant clinical pregnancy results in patients with azoospermia.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.195-200
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• 2015

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female $F-{_1}$ mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG ($81.1{\pm}15.1$), 1,2-PROH ($88.0{\pm}21.1$) or the control ($99.9{\pm}21.3$) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO ($14.7{\pm}1.3$), EG ($12.1{\pm}1.1$), Glycerol ($15.2{\pm}1.8$), and 1,2-PROH ($11.5{\pm}1.3$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.5{\pm}0.7$, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

• Development and Reproduction
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• v.15 no.4
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• pp.385-391
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• 2011

Human in vitro fertilization and embryo transfer (IVF-ET) program is a general procedure for infertile couples since first successful delivery on 1978 in UK and Korean first on 1985. Recently in Korea, more than 42,000 cases per year of IVF-ET were performed and showed good pregnancy rates compared worldwide data. The human IVF-ET procedure use many consumables, such as ovum pick-up (OPU) needles, centrifuge tubes, culture dish, ICSI pipette, culture media and ET catheters. Major of these materials are supported by the global companies. Thus, Korean IVF-ET program might be placed unstable situation by global economical risks. These uncertain problems could be overcome by the domestic production of IVF-ET materials and consumables. Two times questionnaires for Korean clinicians and researchers about the domestic production were performed and analyzed. Many of them requested domestic OPU needles, ET catheters, culture media and ICSI pipettes under good quality control and quality assurance system. This trial may be contributed to industrialization and to global competence of Korean IVF-ET program. The results of this survey can be provide a fundamental base for development and production of domestic materials and consumables for human IVF-ET program.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.35-40
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• 2006

This study was performed to investigate the effects of embryo developmental stage and superoxide dismutase (SOD) on the survival of frozen-thawed porcine embryos by open pulled straw(OPS) method. Porcine IVF blastocysts were frozen-thawed by OPS method and cultured for 48 h under the existence of SOD. There are no significant differences in the proportions of normal morphology among the early, mid- and expanded blastoryst stages $(30.8{\sim}38.6%)$. After culture of embryos, the developmental rates to the expanded blastocyst stage(38.7%) were significantly higher than those of other stages (P<0.05). The proportions of expanded and hatched embryos were higher in medium with 1 unit/ml SOD than 0 and 10 units/ml of SOD. The result indicates that OPS method can use for the pig embryo cryopreservation, especially for the late stage blastocysts. SOD may can reduce the demage of frozen-thawed porcine embryos.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.11-20
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• 1997

The influence of cryopreservation of donor embryos on the in vitro developmental potential in the nuclear transplant rabbit embryos was evaluated. The embryos of 16-cell stage were collected and cryopreserved with EFS solution by vitrification method. The frozen embryos were thawed and synchronized to S and G$_1$ phase of 32-cell stage. The recipient/ cytoplasms were obtained by removing the first polar body and chromosome mass from the oocytes collected by non-disruptive microsurgery procedure. The separated S and G$_1$ phase blastomeres of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20 hrs post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells. After in vitro culture for 120 hrs, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. The electrofusion rate was significantly (P<0.05) reduced in the frozen nuclear donor,compared with fresh donor nuclei as 80.0 vs 62.8% in S phase and 81.7 vs 64.8% in G$_1$phase, respectivley. The in vitro developmental rate to blastocyst stage with the S and G$_1$phase of fresh embryos(26.3 and 61.1%, respectively) was found significantly (P<0.05) higher, compared to the S and G]phase of frozen embryos(11.9 and 34.6%, respectively). When frozen as well as fresh donor embryos were synchronized to G$_1$ phase, the in vitro developmental rate to blastocyst stage was significantly (P<0.05) higher, compared with S phase donor nuclei. The cell counts of nuclear transplant embryos developed to blastosyst stage were significantly (P<0.05) more in G$_1$ phase of fresh or frozen embryos (180.1 and 125.7 cells, respectively), compared with S phase nuclear donor (145.1 and 103.7 cells, respectively). From the above results it was concluded that the rabbit embryos cryo- preserved by vitrification might be available as nuclear donor, though the developmentalpotential and cell counts of nuclear transplant rabbit embryos were decreased significantly.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.235-241
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• 2017

To preserve genetic materials, cryopreservation of the semen from live animals is the main technique to establish cryo-banking system which could be used for artificial insemination and embryo transfer. However, the population of Korean black goat (KBG) becomes to dwindle in number and is now faced genetic erosion by crossbreeding with non-native breeds in small KBG farms. In this study, simple freezing method was used to preserve frozen semen from KBG using spermatozoa of cauda epididymis (CE) and electro-stimulated semen (ES). The negative effects of seminal plasma on fresh sperm was confirmed using precipitation test of Triladyl egg yolk diluent and sperm viability after thawing was compared between CE and ES spermatozoa. When seminal plasma of fresh ES semen was washed with semen washing media (SWM), the rates of live sperm shown no significant difference between CE and ES spermatozoa before freezing. However, the survival rate of frozen/thawed CE sperm was higher than ES ($74.6{\pm}10.6%$ vs $53.8{\pm}5.2%$) with significant difference (p < 0.05). The results of longevity test on frozen/thawed sperm from CE showed healthier sperm than ES. Therefore, spermatozoa from CE could be used for cryo-banking system in KBG lines. The more studies are needed to increase survival rate of ES semen.

• Development and Reproduction
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• v.4 no.2
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• pp.161-173
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• 2000

The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.