• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.311-316
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• 1993

Bovine follicular oocytes were matured, fertilized and cultured in vitro. Oocytes with or without cumulus cells were transferred into TC-I99 medium supplemented with 10% FCS 8 h after insemination, and embryos cleaved to 2- to 8-cell stage 56 h after insemination were cultured further for 5 days. The proportions of embryos developed to morular and blastocyst stages were significantly higher (P<0.0l) in those cultured with(31%) than without(15%) cumulus cells. When the embryos were cultured with cumulus cells in the medium with different protein sources, the highest proportion(59%) of embryos developed to morular or blastocyst stage was obtained in medium with BSA(P<0. O01). Higher proportions of embryos were degenerated during culture with FCS(15%) or CS(14%) compared with BSA(3%). The present results indicate the early development of in-vitro fertilized bovine embryos can be maintained efficiently by BSA when they were co-cultured with cumulus cells.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.95-102
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• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.103-109
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• 1997

The studies on the carried out to investigate to determine the optimum thawing temperature and equilibration time and 1 step straw method of frozen bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1 $\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as followes : 1. The equilibration time on in vitro developmental rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20 min.). 2. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher in vitro developmental rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$. 3. The thawing time on in vitro developmental rates of bovine embryos was attained after short period of time(1~5 min.) in the freezing mediuim higher than long period of time(10min.). 4. The in vitro developmental rates of bovine embryos after rapid frozen-thawing by 1 step straw method in the freezing medium added 1.5M, 2.0M glycerol, DMSO, propanediol and 0.25M, 0.50M, 0.75M, 1.00M sucrose were 12.5~19.4%, 10.0~15.6%, 9.1~13.8% and 6.7~12.9%, respectively.

• Journal of Animal Science and Technology
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• v.47 no.3
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• pp.363-370
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• 2005

Experiments were conducted to determine the effects of beta-mercaptoethanol ($\beta$-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and $\beta$-ME (0, 25, 50 and 100 ${\mu}$M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 ${\mu}$M $\beta$-ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing $\beta$-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.123-128
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• 2011

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.4 no.2
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• pp.151-156
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• 1991

Bovine in vitro fertilization experiment was carried out using ovary-derived follicular oocytes and frozen-thawed spermatozoa to determine the optimal preincubation time of spermatozoa and the insemination time for successful in vitro fertilization rate. The possibility of parthenogenetic cell division of unfertilized oocytes during culture without spermatozoa was also examined. There was no significant (p>0.05) difference in percent ratio of embryos developed to blastocyst stage between 0 and 3 h preincubation times of spermatozoa, showing a tendency to have higher percentage for 0 h of preincubation time. The 6 h insemination time seemed to be better for producing higher percentage of ova cleavage compared with those of 1 and 3 h treatments. Approximately 10% of unfertilized oocytes divided into 2 to 4-cell stage, and some of them cleaved to 5 up to 8-cells. The results obtained from this study suggested that 0 h of sperm preincubation time and 6 h of insemination time would be suitable for producing better in vitro fertilization rate of bovine oocytes. It is also likely that unfertilized bovine oocytes probably cleave to some cell stages with irregular divisions of the cells. On the one hand, considerable variation was also found in spermatozoa function among individual bulls.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Animal cells and systems
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• v.10 no.4
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• pp.211-217
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• 2006

Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01-100 ${\mu}M$), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH-induced pregnenolone ($P_5$) production by the follicles ($EC_{50},\;4.0{\mu}M,\;25.6{\mu}M\;and\;5.7{\mu}M$, respectively ), and also suppressed the conversion of testosterone (T) to estradiol $17{beta}\;(E_2)\;(EC_{50},\;4.2{\mu}M,\;7.5{\mu}M\;and\;80.0{\mu}M) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as $C_{17-20}$ lyase and $17{\beta}$-hydroxysteroid dehydrogenase ($17{\beta}$-HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.185-193
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• 1999

To improve the efficiency of in vitro production of embryos with follicular oocytes in pig, the recovery rates, in vitro fertilization and development. The results obtained were as fellows: The number of oocytes recovered 37 ovary was 1,365 by aspiration, 1,884 by slicing and 3,830 aspiration post slicing, per ovary was averaged 103.5 aspiration post slicing than 30.7 by aspiration and 50.8 by slicing (P<0.05). The percentage of grade I and II oocytes recovered was 0.05∼0.2% and 1.7∼2.3% respectively(p<0.05). The fertilization rates of ejaculate and epididymis sperm was 83.0 and 83.1%. And cleavaged rate was 60.8 and 69.0% respectively(P<0.05). However, there were no significant differences between sperm sources. The clevage rates of fertilized oocyte was significantly(P<0.05) higher as B.O(92.8%) than TALP (90.1%) or mTBM (80.1%). And in vitro developed to blastocyst rates of mTBM media used for fertilization was significantly (P<0.05) higher as 12.4%, compared with the results using the media of TALP(1.6%) or B.O (0.0%). The embryos developed 2-cell stage after in vitro fertilization were co-cultured with or without POEC and BOEC in NCSU-23 and TCM-199 media. In vitro developed to blastocyst rates was NCSU-23 with POEC(2.3%) or BOEC(1.2%), but in vitro cultured in TCM-199 medium with POEC or BOEC was not developed to blastocyst. The percentage of embryos that developed to morula stage in 0, 50, 100, 200 and 250uM was 16.6, 22.0, 13.5, 19.0 and 22.0%, respectively.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.