• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.157-162
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• 2006

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs (cumulus oocytes complexes) in vitro. Attempts had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of FGF (fibroblast growth factor) on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in HPM 199 (Inst. of Functional peptide, Japan) containing 0.1, 1 and 10 ng/ml FGF for 24 hr, maturation rates to metaphase II ($70.0{\sim}75.0%$) were significantly higher (p<0.05) than that of control group (0 ng/ml FGF, 37.5%). When matured COCs with FGF were cultured in maturation medium after in vitro fertilization, developmental rates to blastocysts were 9.5, 0 and 2.9%, respectively, compared to 25.0% of the control group (p<0.05). When the matured COCs with FGF were cultured in HPM 199 (IFP971, Inst. of Functional peptide, Japan) containing 10% FBS, 0.8% BSA or 0.1% PVA (polyvinyl alcohol), the blastocyst formation rates were 12.4, 12.8 and 8.5%, respectively, while the rates of matured COCs with FGF and cultured with IVMD and IVD (Inst. of Functional peptide, Japan) without serum were 38.4% and 34.8%, respectively (p<0.05). These results suggested that FGF is available for in vitro maturation of bovine COCs and is not suitable for in vitro development, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine embryo development.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.89-95
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• 2000

This study was to establish an effective dilution technique in a vitrification of bovine blastocysts for the field trial. For vitrification, blastocysts were exposed in glycerol (G) and ethylene glycol (EG) mixture in m-DPBS supplemented with 10% FBS. Blastocysts were first exposed to 10% (v/v) G for 5 min, and subsequently were transferred to 10% G plus 20% EG (v/v) for 5 min. Finally, embryos were transferred to 25% G plus 25% EG (v/v) for 30 see and were placed in nitrogen vapor for 3 min, and then were plunged into L$N_2$. At thawing, the straw containing blastocysts was placed in air for 10 sec, and then plunged into a water bath at $25^{\circ}C$ until all ice had disappeared. They were placed in $25^{\circ}C$ and 36$^{\circ}C$ water according to treatment group for different time. Also, in vitro survival was assessed by the re-expansion and hatched rates at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) In the survival rates of vitrified bovine blastocysts according to different dilution time at thawing, the data of 1 min group (86.6, 56.6%) were higher than those of other treatment groups (2 min; 93.5, 35.4%, 2.5 min; 76.9, 30.7%, 3 min; 88.8, 36.1% and 3.5 min; 83.7, 8.1%). 2) When the in vitro survival of vitrified groups according to different developmental stage was examined at 48 h after thawing using 1 min dilution method, the hatching rates of fast developed embryos (expanded blastocyst: 81.3%: early hatching blastocyst: 86.2%) were higher than that of delayed developed one (early blastocyst: 46.6%). 3) In addition, when the in vitro survival of vitrified groups according to different embryo age was compared, the hatched rates at 48 h after thawing of Day 7 (66.6%) and Day 8 embryos (60.0%) were significantly higher than that of Day 9 embryos (22.7%) (P＜0.05). These results demonstrate that vitrified bovine IVM/IVF/IVC blastocysts can be successfully survived in vitro using one-step dilution (1 min) method.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.181-185
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• 2004

This study was conducted to examine whether activation treatments, source of oocytes and culture conditions affect in vitro developmental ability of caprine oocytes. Mature Korean native goats were pretreated with intravaginal CIDR for 10 days. The goats were then treated with a single intramuscular injection of 1,000 IU PMSG on Day 8 or twice daily injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR insertion for superovulation. All the goats were injected with 10 mg PGF/sub 2a/ on Day 8 and 400 IU hCG on Day 10 of CIDR. Oocytes were surgically collected by oviduct flushing(in vivo maturation) or direct follicle aspiration(in vitro maturation) through mid-ventral incision at 35 h after hCG injection. Fifteen to twenty oocytes were placed in TCM-199 medium containing 25 mM Hepes and hormones under mineral oil at 39℃ in a humudified atmosphere of 5% CO₂ in air for 22 to 24 h. After maturation, the oocytes were activated by electric stimulation or ionomycin + 6-DMAP. The activated oocytes were then cultured in M16, TCM-199 and mSOF media supplemented with proteins at 39℃ for 6 to 7 days. Activation treatments did not affect cleavage of the oocytes. The cleavage rates were 64.1% (41/64) in oocytes activated by electric stimulation and 76.5% (218/285) in oocytes activated by ionomycin + 6-DMAP. The proportion of development to blastocyst was 15.6% (34/218) in oocytes activated by ionomycin + 6-DMAP, but activation by electric stimulation did not support embryos developed beyond morula stage. There were no differences in the cleavage rates of activated oocytes experiencing in vivo (86.8%, 66/76) and in vitro maturation (69.0%, 127/184). However, the development rate to blastocyst stage was significantly (P<0.05) higher for oocytes matured in vivo (50.0%, 33/66) compared to in vitro (0.8%, 1/127). Culture conditions did not affect the cleavage of -activated oocytes. The cleavage rates were 51.6% (49/95) in M16, 64.3% (18/28) in TCM-199 and 81.0% (145/179) in mSOF, respectively. By contrast, the development rate of activated oocytes to stage was greater (P<0.05) for oocytes cultured in mSOF medium (23.4%, 34/145) than in M16 or TCM-199 (0.0%). Our results suggest that source of oocytes and culture conditions are major factors affecting in vitro development of caprine parthenogenetic oocytes.

• Development and Reproduction
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• v.16 no.2
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• pp.137-143
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• 2012

The main objective of this study is to investigate the effects of ovulation induction by treating HCG, LHRHa, GnRHa, ovaprim and pimozidein in long snout bullhead, L. longriostris. All hormons were injected into the muscles of back. Concentration of LHRHa to injection were 20, 50, 100, 150 and 200 ${\mu}g/kg$ and same concentration of LHRHa was injected after 24 hour. The ovulation induction rate was 100% and fertilization and hatching rates were 68.4, 45.2, 58.4 and 33.6% in 50 and 100 ${\mu}g/kg$. The times to ovulation were between 28 and 44 h. HCG was injected in long snout bullhead at 5,000, 10,000, 15,000, 20,000 and 25,000 IU/kg. The ovulation induction rate was 50% in 15,000 and 20,000 IU/kg. Fertlilzation and hatching rates were 55.2, 45.6, 52.8 and 44.8%. Ovulation time was between 72~80 h. HCG concentration of 500, 1,000, 2,000 and 4,000 IU/kg were injected with $50{\mu}g$ of LHRHa. Ovulation and hatching rates in 2,000 IU/kg were 75 and 35%. Ovulation time was 28~48 h. Ovaprim of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL/kg were injected to the abdominal cavity. The ovulation induction rate was highest at 2.5 mL/kg to 50% and ovulation time was between 66~86 h. LHRHa concentration of 50, 100, 200, 300 and 400 ${\mu}g/kg$ was injected with pimozide (1,000 ${\mu}g/kg$). Ovulation induction rate was 87% at 100 and 200 IU/kg with pimozide. Ovulation time was between 66~86 h.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.61-68
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• 2002

The present study was examined effects of aesculetin and $O_2$concentrations on in vitro development of Hanwoo (Korean Native Cattle) embryos derived from in-vitro matured and fertilized (IVM-lVF) oocytes. The oocytes were cultured for the first 40~44 h after in vitro fertilization, then embryos of 2 to 8 cell stages were cultured under the different culture conditions for another 6 days. In experiment 1, the higher rates of morulae and blastocysts were produced in 5% $O_2$, than in 20% $O_2$(P＜0.05). There was significantly (P＜0.05) higher in embryos cultured with 1 $\mu\textrm{g}$/$m\ell$ than with 0, 5 and 10 $\mu\textrm{g}$/$m\ell$ of aesculetin. In experiment 2, the proportions of embryo developed with blastocysts and morulae plus blastocysts in 5% $O_2$, again was significantly (P＜0.05) higher in 20% $O_2$, during the culture with aesculetin and/or taurine. In the 5 and 20% $O_2$atmosphere, the inclusion of 1 $\mu\textrm{g}$/$m\ell$ aesculetin or 2.5mM taurine increased significantly (P＜0.05) the percentages of blastocysts and morulae plus blastocysts. In experiment 3, in medium with aesculetin plus PDGF and taurine plus EGF than other treatment groups, significantly (P＜0.05) higher developmental rates were obtained. Number of blastomere in balstocyst stage were also higher in medium with that than without aesculetin. However, there were no significant differences in all culture conditions. In experiment 4, the proportions of embryo developed to the morulae and blastocyst stages were significantly (P＜0.05) higher rates in medium with natural and commercial aesculetin than in control medium. No significant differences, however, were observed in between natural (71%) and commercial (70.0%) aesculetin. Number of blastomere in blastocyst stage were also higher in medium with natural and commercial aesculetin than in control medium. However, there was no effect on the number of blastomeres by these treatment. These data indicate that preimplatation embryos are very sensitive to condition that can cause oxygen concentration and show that efficiency role of aesculetin for improving bovine embryo development in vitro.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.101-107
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• 2006

The objective of this study was to investigative the effects of retinol supplement to IVM and/or IVC medium on maturation, fertilization and development of pig oocytes. North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF) was used as base medium. Each 1 uM, 5 uM and 10 uM concentration of retinol was supplemented to IVM and /or IVC medium. When the retinol was supplemented to maturation medium, the maturation rates were not different (p>0.05) among treatment groups ($66.7{\pm}6.0{\sim}69.2{\pm}5.3%$), but the developmental rate to blastocyst stage was higher (p<0.05) in $5{\mu}M$ group ($20.4{\pm}2.6%$) than in 0 uM ($13.6{\pm}2.1%$) and 10 uM groups ($9.7{\pm}1.7%$). Moreover, total cell number was significantly greater (p<0.05) in the 5 uM group ($37.0{\pm}1.6$) than in the other groups ($29.8{\pm}1.0{\sim}33.2{\pm}1.0$). Retinol supplement to maturation medium did not significantly affect the rates of fertilization and polyspermy (p>0.05). When the retinol was supplemented to culture medium or both maturation and culture medium, the rates of cleavage, and develop to morula and blastocyst stage were not affected, while those of 10 uM group were significantly decreased (p<0.05). These results indicate that 5 uM retinol supplement in maturation medium significantly stimulates embryo development, also improves the total cell number of blastocyst stage in pig.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.119-126
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• 2004

The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.121-126
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• 2007

The objective of this study was carried out to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as non-invasive marker to know the developmental competence in advance. The porcine oocytes matured for 48 h were examined the polar body extrusion. The examined oocytes were matured for additional $16{\sim}18h$ and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 h for diploid formation. The treated oocytes were cultured and examined the cleavage after 48 h and continued culturing for 5 days. The oocytes of 21.9% were discarded in morphological selection and 32.1% oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated and then after 48 h the cleavage rates were examined. In morphologically selected oocytes, 15.8% oocytes were not cleaved and 52.6% oocytes were normally cleaved and 31.6% oocytes were hyper-cleaved over 8-cell stage. However in the first polar body extruded oocytes, 7.1% oocytes were not cleaved and 73.1% oocytes were normally cleaved and 19.8% oocytes were hyper-cleaved. The morphologically selected embryos that not cleavage-selected were developed in 16.7% up to blastocyst and the morphologically selected and cleavage-selected embryos were developed in 31.7%. The polar body extruded oocytes that were not carried out cleavage selection were developed in 39.0% and the polar body extruded and cleavage-selected embryos were developed 49.0%. The first cleavage timing was examined with 12 h interval after activation. In $0{\sim}12,\;12{\sim}24,\;24{\sim}36,\;and\;36{\sim}48h$ intervals, 4.1%, 68.6%, 19.1%, and 2.3% oocytes were cleaved and 5.9% oocytes were not cleaved until 48 after activation. The cleaved oocytes in each interval were cultured and developed upto blastocyst with 0, 39.1, 9.5, and 0%, respectively. This results suggests that polar body extruded and cleaved at $12{\sim}36h$ embryo has higher developmental potential than the others.