• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.110-110
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• 2003

The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for IVF involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Sperm pellet was prepared in the tube and mature oocytes were placed on cell strainer with $70 \mu m$ pore size (Falcon 2350) at the top of the tube. After insemination, the oocytes were stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While having similar penetration rates in both methods ($86.67 \pm 2.36% to 83.33 \pm 1.36%$), there was a significant reduction of polyspermy in modified swim-up method ($17.50 \pm 1.60%$) compare to the control ($44.1 \pm 3.70%$ (p<0.05). Subsequent culture showed higher rate of blastocyst formation in modified swim-up method (20.44$\pm$0.99%) than the control ($15.73 \pm 3.26%$) (P<0.05), even though there was no significant difference. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure required to implicate this method in routine porcine IVF.

• 한국가축번식학회지
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• 제24권3호
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• pp.311-317
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• 2000

본 연구는 체외에서 한우 난포란의 성숙과 수정후 배의 체외발생에 있어서 온도충격의 영향이 처리온도 및 처리시간에 미치는 효과를 검토하였다. 1. 체외에서 생산된 4~8 세포기 배의 배반포로의 배발달율에 있어서 온도충격의 적정온도는 41$^{\circ}C$, 처리시간은 30초였다. 2. 체외성숙 후의 온도충격이 체외성숙 전의 온도충격보다 수정란의 분할율을 유의하게 상승시켰다(p<0.05). 3. 체외성숙 후와 배양 5일째 배에 각각 온도처리를 하였을 경우 비처리군보다 배반포의 배발달율이 향상되었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.39-39
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• 2002

The mechanisms underlying the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear. It is known that in vitro produced embryos show more frequent occurrence of fragmentation, which result in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in in vitro fertilization (IVF) and nuclear transferred (NT)bovine blastocyst. (omitted)

• 한국수정란이식학회지
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• 제10권2호
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• pp.131-138
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• 1995

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 $\mu$g /mL of FSH and LH and 1 $\mu$g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

• 한국가축번식학회지
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• 제27권1호
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• pp.35-44
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• 2003

The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

• 대한수의학회지
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• 제33권3호
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• pp.547-554
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• 1993

This study was carried out to investigate the best condition for in vitro and in vivo culture after freezing and thawing of nuclear transplant 2 cell embryos. When nuclear transplant embryos were submitted to electrofusion, the significantly higher fusion rates of 2 cell donor nuclei were achieved at the electric field strength of DC 1.5 kV/cm for 100 and $150{\mu}sec$, DC 2.0 kV/cm for 100 and $150{\mu}sec$ than DC 1.0 kV/cm for 100 and $150{\mu}sec$(p<0.01). The significantly higher fusion rates of 4 cell donor nuclei were achievecl at DC 2.0 kV/cm for 100 and $150{\mu}sec$ than DC 1.0 kV/cm for 100 and $150{\mu}sec$(p<0.01). The fusion rates in 8 cell donor nuclei were 94.2~99.3%. The developmental potency to blastocyst in 2 cell donor nuclei was significantly higher in DC 2.0 kV/cm for $150{\mu}sec$ treated group(p<0.01). The significantly higher developmental potency to blastocyst in 4 cell donor nuclei were achieved at the electric field strength of DC 2.0 kV/cm for $150{\mu}sec$ than DC 1.5 kV/cm for 100 and $150{\mu}sec$, DC 2.0 kV/cm for $100{\mu}sec$ treated group(p<0.01). The develop mental potency to blastocyst in 8 cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}sec$ treated group(p<0.01). The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2 cell donor nuclei than in 8 cell donor nuclei(p<0.01). When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant(p<0.01). The production rates of offspring after transfer of nuclear transplant embryos to recipient mouse were no significant difference in 2, 4 and 8 cell donor nuclei.

• 한국가축번식학회지
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• 제21권3호
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• 한국수정란이식학회지
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• 제12권3호
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.207-212
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• 2005

In the present study, we investigated the effects of genotypes on in vitro maturation and fertilization in porcine fresh/frozen-thawed oocytes. The porcine cumulus-oocyte complexes (COCs) were divided into four groups according to whether they were: (1) in vitro matured; (2) cryopreserved and in vitro matured; (3) in vitro fertilized and (4) cryopreserved, and in vitro fertilized. Maturation of porcine COCs was accomplished by incubation in NCSU23 medium. Immature oocytes were cryopreserved by Open Pulled Straws (OPS) method according to Vajta et al., (1998). Oocytes stained by Acetic-Orcein method were observed under the microscope. DNA extracted from the ovaries was analyzed by RAPD (random amplified polymorphic DNA) and SSCP (single strand conformational polymorphisrrt) method. The rates of oocytes maturation and fertilization were significantly high in AA genotype. The results indicated that in vitro maturation and fertilization in porcine fresh/frozen-thawed oocytes may be affected by genotypes in pigs.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.173-181
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• 2012

Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with $1{\mu}M$ of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.