• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.237-243
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• 2008

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25-ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen-thawed boar spermatozoa for 6h in a modified tris-buffered medium (mTBM) or in its modified medium by substituting the tris with 25mM sodium bicarbonate (modified bicarbonate-buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University-23 medium for embryo development. The percentage of live sperm was $47{\pm}4%$ and morphological abnormality of acrosome was found in $14{\pm}3%$ of spermatozoa. Optimal sperm concentration for IVF was $0.75{\sim}1.0{\times}1.0{\times}10^6$ sperms/ml when mTBM containing 5mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1mM caffeine than from those fertilized in mTBM with 5mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.

• Korean Journal of Agricultural Science
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• v.22 no.1
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• pp.62-68
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• 1995

This study was carried out to examine splitting, developmental capacity and rapid freezing of blastomeres separated from 2-, 4-, 8-cell and morula from porcine embryos. The results obtained in this study were summerized as follows : 1. The successful splitting rate by pronase was 85.7% in 2-cell embryos(average splitting rate, 68.0%), and by manipulator was 76.6% and 74.3% in 2- and 4-cell embryos. 2. The developmental capacity rates of splitted embryos by the pronase treatment were 24.1%, 20.4%. 25.5% and 26.6% in 2-, 4-, 8-cell and morula, and by manipulator were 36.4%, 39.5%, 36.1% and 41.9%, respectively. 3. The successful results of in vitro culture after frozen-thawed of splitted embryos were 16.1%(glycerol) in 2-cell, 16.7%(DMSO) in 4-cell and 27.6%(ethyleneglycol) in morula, respectively.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.41-48
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• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• Journal of Veterinary Clinics
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• v.9 no.1
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.245-250
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• 2001

This study was performed to evaluate whether vitrification method using ethyle glycol and eletron microscopic (EM) grid could be used far the cryopreservation of human oocytes in ART program. Surplus oocytes were obtained from consented IVF patients. These surplus human oocytes were frozen with our vitrification method, Oocytes were exposed to 1.5M ethylene glycol (EG) in DPBS far 2,5 minutes, followed by 5.5M EG plus 1.0M Sucrose in DPBS for 20 seconds. Then oocytes were transferred onto the EM grid and the grid was plunged into LN2 for storage. For thawing, oocytes containing EM grid were sequentially transferred in 1.0M, 0.5M, 0.25M, 0.125M and 0 M sucrose in DPBS solution at the intervals of 2.5 minutes. Thawed and survived oocytes were provided for ICSI. Embryos from vitrified oocytes were transferred to uterus of the patient on 4 to 5 days after ovulation in natural cycles of on 15 to 17 day of hormone replacement cycles. A total of 370 oocytes from 26 patients were thawed and 159 (43.0%) of them survived. One hundred thirty four oocytes (84.3%) were fertilized normally and 126 pre-embryos were transferred to 26 patients, resulting in 5 clinical pregnancies. The pregnancy rate per transfer was 19.2% and implantation rate was 4.0%. Among the five pregnant, 4 patients delivered 4 healthy babies and the one patient was 32-week ongoing pregnancy. From this results, vitrification using ethylene glycol as cryoprotectant and EM grid is a rapid and simple method that can be effectively applied for the cryopreservation of human oocytes in ART program.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.97-108
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• 2003

In-vitro fertilized Hanwoo embryos were biopsied for sex determination by PCR. Biopsied embryos were incubated for 1∼2 hours for the recovery. Those sexed Hanwoo embryos were transferred to 49 Hanwoo and 16 Holstein recipients from February 2000 to February 2001. Of 65 recipients, 14 cows(12 Hanwoo and 2 Holstein) delivered the same offspring as sex-determined by PCR, therefore the conception rate was 21.5%. 1. Total 65 embryos(male 35, female 30) were transferred to recipients, and 14 calves (male 6, female 8) were delivered. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex determination was 100.0%. 2. The conception rate after transfer with biopsied embryo between Hanwoo and Holstein was 24.5% and 12.5% 3. The conception rate after transfer with biopsied embryo between fresh and frozen-thawed embryos was 23.5% and 14.3%. 4. The conception rate according to the season when embryo was transferred was 11.8, 29.4, 23.5 and 20.0% for spring, summer, autumn and winter, respectively. 5. The conception rate according to embryo quality after biopsy was 41.7, 30.0 and 0.0% for excellent, good and fair quality. 6. The conception rate according to thickness of uterine horn was 71.4, 18.9, 11.8 and 0.0% for 0, +, ++ and +++ thickness. 7. The conception rate according to the site in the uterine hem where embryo was put was 30.0, 20.0 and 10.0% for cranial, mid, and caudal part of uterine horn. 8. The conception rate according to the quality of corpus luteum ipsilateral to the uterine horn where embryos was transferred was 41.2, 14.3 and 15.4% for excellent, good and fair quality. 9. The conception rate according to the time required for embryo transfer was 18.2, 30.0, 30.0, 0.0 and 25.0% for 10, 15, 20, 25 and 30 minutes. 10. The conception rate according to parity of recipients was 26.5, 19.1, 14.3 and 0.0% for the primiparous, the 2nd parous, the 3rd parous and the 4th parous recipients. These results indicated that fresh embryos are more demanded than frozen-thawed embryos for good conception rate in embryo transfer with biopsied-sexed embryo. Also, it was indicated that we should consider embryo-recovering condition, recipient's uterine thickness, transfer site in uterine horn, quality of corpus luteum, time required for transfer and parity of recipient to achieve good conception rate in ET with biopsied-sexed embryos.