• Journal of Embryo Transfer
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• v.15 no.1
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• Korean Journal of Veterinary Research
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• v.35 no.1
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• pp.187-195
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• 1995

The present study carried out to determine the developmental capacity of bovine oocytes matured in epidermal growth factor(EGF)-containing medium, the developmental competence of bovine embryos using synthetic oviduct fluid(SOF) and the effect of glucose on the development of bovine embryos. In experiment 1, oocytes, obtained from abattoir ovaries, were matured in EGF-containing medium for 24 hours, followed by exposure to Korean native cattle spermatozoa for 18 hours and cultured by utilizing co-culture system with bovine oviduct epithelial cells(BOEC) in TCM199. In experiment 2, early bovine embryos were cultured in SOF with or without BOEC and compared with those in TCM199 with BOEC. In experiment 3, bovine embryos were cultured in the presence or absence of glucose. Seven and ten days after in vitro fertilization, developmental competence of embryos were evaluated. The rate of cleavage was significantly(P<0.05) higher in EGF-containing maturation medium(70.0%) than in control(57.7%). The rates of development to morulae and blastocysts were 30.6% and 23.3% there was no significant difference between them. The rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC(30.4%) and in TCM199 with BOEC(38.0%) were significantly(P<0.01) higher than cultured in SOF without BOEC(13.4%) at seven days after in vitro fertilization. The rates of embryos to blastocysts cultured in SOF with BOEC(29.4%) and in TCM199 with BOEC(35.9%) were significantly(P<0.05) higher than cultured in SOF without BOEC(13.4%) at ten days after in vitro fertilization. The rates of early embryos to morulae and blastocysts cultured in the presence or absence of glucose were 12.2% and 17.5% each other, there was no significant difference between them. The results show that bovine oocytes matured in the presence of EGF can cleave better, SOF with BOEC can replace serum containing complex media, TCM199 with BOEC in bovine embryo culture and glucose have little effect on the culture of early bovine embryos.

• Korean Journal of Agricultural Science
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• v.34 no.1
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• pp.13-18
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• 2007

This study was carried out to investigate the effects of ovarian estrus stage of oocytes and supplementation of medium with BSA, cysteine and myoinositol on in vitro maturation of canine oocytes. The in vitro maturation(IVM) rate to metaphase II (M II) stage of oocytes recovered from different stage of the reproductive cycle (inactive, follicular and luteal stage) cultured in TCM-199 media were $0.0{\pm}0.0%$, $10.0{\pm}4.1%$ and $5.7{\pm}1.6%$, respectively. The IVM rate of oocytes collected from follicular stage was significantly higher in inactive or luteal stage of oocytes. The IVM rates of oocytes recovered from different stage of the reproductive cycle cultured in TCM-199 media with 5% BSA and 0.1 mM cysteine were$0.0{\pm}0.0%$, $15.8{\pm}4.7%$, $5.6{\pm}1.5%$, respectively. The IVM rates of oocytes recovered from different stage of the reproductive cycle cultured in TCM-199 media with 5% BSA and 10 mM myoinositol were $0.0{\pm}0.0%$, $18.4{\pm}4.6%$ and $5.7{\pm}1.9%$, respectively. The IVM rate of follicular stage oocytes was significantly higher in oocytes collected from follicular stage and with cultured 5% BSA and 0.1 mM cysteine or 5% BSA and 10 mM myoinositol compared to other experimental group.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.111-117
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• 1996

This experiment was designed to evaluate the effect of transforming growth factor-$\beta$ (TGF-$\beta$) and insulin-like growth factor-I (IGF-I) in bovine oocyte maturation in the presence or absence of serum on subsequent fertilization and embryo development. In addition, various concent rations of these growth factors were evaluated for the ability to promote development of eight-cell stage embryos to the blastocyst stage. Cumulus-oocyte complexes were recovered from 2 to 6 mm follicles obtained from slaughterhouse ovaries and cultured at 38.5$^{\circ}C$ for 24 hours in TCM-199 (HEPES Modification) with or with out 20 % fetal bovine serum (FBS) to which the following growth factors were added TGF$\beta$ IGF-l or TGF $\beta$ + IGF-I, all at 10 ng/ml each. The matured oocytes were fertilized in IVF-TL medium with frozen-thawed semen at a concentration of 1 ${\times}$ 10$^6$ cells/ml of fertilization medium following Percoll separation. After 24 hours of sperm-egg incubation, the embryos were transferred to CZB medium without glucose for 48 hours and then cultured in TCM-199 with 20% fetal bovine serum (FBS) for 96 hours. The addition of growth factors to IVM medium in the presence of serum had no effect on cleavage and subsequent embryo devlopment to blastocyst. In the absence of serum, TGF- improved cleavage and development to blastocyst compared to control's(p<0.05) and no synergistic effeet of IGF-I + TGF-$\beta$ was observed. In the second experiment, eight-cell embryos obtained by in vitro maturation (IVM) in TCM-199 + 20% FBS without growth facrors and in vitro fertil-ization (IVF) were cultured in the in vitro cuiture (IVC) medium supplemented with 5, 10 ng/ml TGF-$\beta$ or 5, 10, 50, 100 ng/ml IGF-I. Cleavage rate and development to the blastocyst stage was observed during seven days of incubation. The supplementation of 10 ng/ml TGF-$\beta$ to lVC medium for eight-cell embryos improved development to blastocyst (p<0.05) compared to control. In conclusion, these data indicate that the supplementation of growth factors to IVM medium in the presence of serum does not influence cleavage and subsequent embryo development. However, significantly more oocytes matured in serum-free TCM-199 and eight-cell embryos cultured in lVC medium developed to blastocyst with supplementation of 10ng/ml TGF-$\beta$.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.269-274
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• 2008

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.137-137
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• 2003

난자의 체외성숙 및 체외배양에는 일반적으로 동물의 혈청을 기본배양액에 5-10% 정도 첨가한 배양액을 사용하고 있다. 그러나, 혈청으로부터 바이러스, 세균, 마이코 플라즈마 등에 오염될 가능성이 있기 때문에, 본 실험에서는 완전 무혈청 배양액에서 난자의 성숙, 배발생, 세포수, 동결성을 검토하였다. 도축된 한우의 난소로부터 채취한 난자는 선별하여 TCM199+10% FBS와 IVMD 101 배양액에서 22~24시간 동안 체외성숙시킨 후, IVF 100(일본, 펩티트연구소)으로 2회 세정한 후, 각각의 배양액 50${\mu}\ell$ 소적에 5개씩 5~6 시간 수정시켰다. 체외수정한 수정란은 TCM 199+10% FBS, IVMD 101, IVD 101 배양액에서 7~8일간 배양하여 배발생율을 조사하였다. 발생된 배반포의 일부는 세포수를 조사하였고 나머지 배반포는 1.8M EG로 동결하였다. (중략)

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.