• Journal of Embryo Transfer
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• v.20 no.3
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• pp.303-309
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• 2005

This study was conducted to examine the effects of OA on metaphase of meiosis II and the mitochondrial activity of cytoplasm in bovine cumulus oocytes complexes(COCs) during in vitro maturation. Hanwoo COCs were collected from the slaughterhouse cow ovaries and matured in TCM199 supplemented with $0.1\%$ PVA, 0.2 uM, 2 uM, 20 uM OA for the maturation rate of OA concentration. For the maturation effects between OA and cycloheximide(CX), COCs were matured in TCM199 with 25 ug/mL CX, 25 ug/mL CX (6 hrs culture) plus 2 uM OA or 2 uM OA only at a atmosphere $5\%\;CO_2,\;95\%$ air $39^{\circ}C$ for 6, 12, 24 hrs. To evaluate the nuclear types of matured COCs, cumulus cells were removedby $0.5\%$ hyaluronidase sol. and oocytes were fixed in 1:3 acetic acid ethyl alcohol for 30 sec. and then stained with $0.1\%$ basic Fuchsin sol. For the detection of fluoriscent intensity (FI) of matures oocytes, cumulus cells were removed same as performed above and were stained with 20 nM mite tracker for 20 min. at $39^{\circ}C$. Mitochondrial activity of FI in matured oocytes was imaged by laser conforcal microscopy (Fluoview, Olympus, Japan) and were measured scanned face on 5 um from median to endpoint of oocytes. Statical analysis of nuclear types observed the three replicates was carried out with ANOVA and Fisher's protected least significant difference test using the STATVIEW program. FI of matures oocytes was compared the multiples of the least intensity among the measured oocytes. Maturing in TCM199 supplemented with $0.1\%$ PVA, 0.2 uM, 2 uM, 20 uM OA, metaphase B were showed 72.0, 50.0, 70.0, $68.8\%$, respectively and there were different significant(p<0.05). In the case of treatment with OA and CX, metaphase were $73.8\%,\;8.2\%,\;45.5\%,\;73.7\%$ in $0.1\%$ PVA-TCM199, 25 ug/mL CX, 25 ug/mL CX plus OA or 2uM OA only, respeclively. FI was revealed the increasing tendency during the process of maturation. Whereas FI in CX was decreased about 3 times compared to the other treatments of 6 hrs maturation. We conclude that OA regulates bovine COCs maturation and induces the mitochondrial activity during the process of maturation.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Development and Reproduction
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• v.1 no.1
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• pp.1-8
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• 1997

본 연구는 단순배양체계내 소 난포란의 성숙에 요구된는 탄수화물 (glucose, lactate와 pyruvate)을 검토하기 위해 수행하였다. GV(germinal vesicle) 단계의 난구세포로 둘러싸인 소 난자를 단백질, 아미노산 및 호르몬이 첨가되지 않은 modified Tyrodes (mT)에서 24시간, 5% $CO_{2}$ 배양기를 이용하여 성숙배양하였다. Glucose 무첨가군 (0-61%)에 비해 5.6mM의 glucose 첨가군 (71-74%)이 유의적으로 높은 (P<0.05) M-II단계로의 난자 발육을 나타내었다. Glucose를 함유한 배지내에서는 lactate(10mM0와 pyruvate(0.5mM)의 첨가에 따르는 M-II 단계로의 발육에 있어 차이를 보이지 않았다. 그러나 glucose 무첨가 배지에서는 pyruvate와 lactate를 첨가하는 것이 첨가하지 않은 것에 비해 condensed GV(76%vs. 0-2%), M-II, (43-61%vs. 0%)단계에 이른 난자수가 유의적으로 높았다. Glucose 함유 mT배지에 lactate와 pyruvate를 첨가하여 난자를 배양하였을 때, 동결융해 정자와 24시간 정치한 후 난자중 87-93%가 정자침투되었고 39-44%가 전핵단계로 발육하였다. 침투난자의 26-30%는 다정자수정이었다. 결론적으로, GV 단계의 소 난포란은 에너지원으로 lactate, glucose와 Pyruvate를 이용하지만, 감수분열 성숙을 유지하는데 glucose가 가장 효과적이다. 이 결과들로 보아 단순배양배지는 소 난포란의 체외성숙에 영향을 미치는 다양한 물질들을 연구하는데 잠재적으로 유용하다.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.139-144
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• 2001

This study was carried out to investigate the effects of the bull, sperm type and sperm pretreatment on the pronuclear formation and in vitro development after injection of spermatozoa into in vitro matured bovine oocytes. 1. Spermatozoa derived room four bulls(A, B, C and D) were used for ICSI. The male pronuclear formation and developmental rates were 73.9∼87.0% and 33.3∼60.9%, respectively. 2. The effects of sperm type were examined. Male pronuclear formation rates by using fresh-and frozen-sperm, tail-cutting and tail-scoring sperm were 82.0%. 78.0%. 42.2% and 51.1% (p<0.05) while development rates were 56.0%. 42.0%, 17.8% and 22.2%, respectively. Fresh sperm achived a high mail pronuclear- and development rates than those of other groups. 3. Cheroical pretreatments were tested and compared. When sperm were pretreated with heparin, BFF(bovine follicula fluid), His, Ca Ionophore(I) and I + caffeine, mate pronuclear formation and developmental rates were 66.7∼82.2% and 33.3∼60.6%. respectively. and these values of treatment of I + caffeine were higher than that of other methods. 4. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated with or without zona pellucida were 80.0%. 72.0% and 46.0%, 36.0%, respectively.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.187-191
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• 2005

This study was designed to investigate the changes of quantity and quality of proteins in medium and cytoplasm during in vitro maturation of bovine oocytes. The total quantity of proteins in medium decreased from 0 to 4.5 hr, but increased from 13.5 to 18 hr after the onset of in vitro maturation. The total quantity of protein in cytoplasm increased from 0 to 4.5 hr, decreased from 4.5 to 9 hr, and increased after 18 hr after the onset of in vitro maturation. A total of 298 protein spots was detected on a gel of 2D SDS-PAGE form maturation medium. Among 28 protein spots expressed significant differences in their quantity, 8 proteins were identified by peptide mass fingerprinting (aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43kDa collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1, transitional endoplasmic reticulum ATPase). Among total of 35 protein spots detected on gel of 2D SDS-PACE from oorytes cytoplasm, $\beta$-tubulin was identified by peptide mass fingerprinting.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.271-281
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• 1996

The effect of superovulation (PMSG, FSH) on the ovarian response of matured cows were tested. The survival rates of bovine embryos and ovarian oocytes frozen by slow, rapid freezing and vitrification were investigated. A total of 15 heads of cow were devided into 3 groups by injection dose of GTH (PSMG, FSH). Each group was superrovulated with injections of 2, 500, 3, OOOJU PSMG and 40mg FSH followed by injection of 30mg PGF2a. Embryos were non-surgically recovered from superovulated cows 6~7days after estrus. The recovered embryos were frozen in 10% glycerol + 10% sucrose by slow and rapid freezing. Ovarian oncytes were frozen in 20% g]ycerol+l0% ethylene glycerol + 30% Ficol + 10% sucrose by vitrification and the survival of frozen embryos and ovarian oncytes were judged by FDA-test. The results are summarized as follows; 1. Estrus after the injection of 2500, 3000 I.U. PMSG and 4Omg FSH were 32.8, 35.0 and 43.4 and the duration of estrus were 18.6, 18.8 and 22.4 hours respectively. 2. The average sizes of the left ovaries were 5.4cm (2, 500 IU PMSG), 5.1cm (3, OOOIU PMSG) and 6.4cm (FSH), and the right were 6.2cm (2, 5001U PMSG), 5.7cm (3, OOOIU PMSG) and 7.&m (FSH) respectively. There were significant differences in the right overies among treatments (P<0.05). 3. The average number of ovarian follicles in the left ovaries were 4.8 (2, 500 IU PMSG), 5.2(3, 000 IU PMSG) and 7.8 (FSH) respectively. There were significant difference in the right ovaries among treatments (P<0.05). 4. In the average numbers of ovulation points in the left ovaries were 3.0 (2, 5001U PMSG), 3.2 (3, OOOIU PMSG) and 4.4 (FSH) respectively, and the right were 7.2 (2, 5001U PMSG), 7.8(3, 000IU PMSG) and 11.4 (FSH). There were significant differences in the right ovaries among treatments (P<0.05). 5. The numbers of the recovered embryos were 20 (2, 5001U PMSG), 19 (3, 000 IU PMSG) and 21 (FSH) respectively, and oncytes and degenerted oncytes were 6.5 and 11.0 Estrus periods of post parturation were 52.4days (2, 5001U PMSG), 69.8days (3, OOOIU PMSG) and 62.4days (FSH) respectively. 6. The FDA score of cow morulae frozen by slow freezing, sernirapid frezing and vitrified freezing was higher in slow (3.1) and vitrified freezing (3.0) than that in semirapid freezing (1.28). The FDA-scores of cow, pig and rabbit ovarian oocytes frozen in 20% glycerol + 10% ethylene glycol + 30% Ficoll + 10% sucrose by vitrification were higher in cows (3.3) than both in pigs (2.6) and rabbits (2.3).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.83-90
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• 1995

Production of calves after transfer of nuclear transplant embryos is the latest technology to be applied in commercial livestock breeding. The objective of this study was to establish an efficient procedure to produce offsprings from nuclear transplant embryos. The fusion rates (72.7% vs. 80.8%), cleavage rates (62.5% vs. 71.4%) and rates of development in vitro (12.0% vs. 15.2%) of nuclear transplant embryos were not significantly different between 30 and 40h maturation age of cytoplast. The in vivo and in vitro-derived embryos as nuclei donor were used in this system of bovine nuclear transplantation. Fusion rates of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (73.0 and 79.2%, respectively). The percentage of embryos reaching the morulae or blastocysts were 21.8% for in vivo-derived embryos and 11.9% for in vitro-derived embryos (p<0.01). Pregnancy rates after embryo transfer of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (45.9 and 40.5%, respectively). However, calving rates after embryo transfer of nuclear transplant embryos were significantly higher in the in vivo-derived embryos than in vitro (p<0.01). Further research for age of cytoplast and use of in vitro-derived embryos as nuclei donor is required in this system. In conclusion, these results clearly show that the use of in vitro-derived oocytes as recipient cytoplast can improve the nuclear transplant system for genetic progress in cattle.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.47-54
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• 1994

The objective of this study was to produce Korean native calves following transfer of in vitro matured, fertilized and cultured embryos into Holstein cows. The ovaries of Korean native cows or heifers were obtained from an abattoir and kept on 25 to 28$^{\circ}C$ and transported to laboratory within 2 hrs. The oocytes were matured in vitro (IVM) for 24 hrs in TCM-199 supplemented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% CO2 in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (IVC) with bovine oviductal epithelial cells for 7 to 9 days. Late morulae and blastocysts produced in vitro were nonsurgically transferred to recipient cows by unilaterial. Recipients were monitored for estrus and for pegnancy by rectal plapation in 60 days after embryo transfer. One of them was pregnant to term and produced a female weighing 42.5kg at birth.