• Journal of Embryo Transfer
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• v.17 no.2
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• pp.129-136
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• 2002

The purpose of this study was to investigate the comparison of viability of bovine blastocysts following glass micropipette (GMP) vitrification and the possibility of production of Korean Native Cow ("Hanwoo,"Bos taurus coreanae) following embryo transfer into Mongolia cows (Bos taurus mongolian). The embryos of Korean Native Cow were produced by IVMFC or superovulation methods in Korea, cryopreserved by GMP vitrification, and subsequently trans-ported to Mongolia. The recipient cows were synchronized using a CIDR plus and prostaglandin $F_2\alpha$($PGF_2\alpha$) treatment. To produce in vivo embryos, seven cows were superovulated using FSH and PGF$_2$／sub $\alpha$／ treatment. A total of 64 blastocysts ( $9.1\pm2.94$ per cow) were collected. In vitro embryos were produced using a defined culture system which cleaved in 80.1％ ova (174／217), and developed to blastocyst stage embryos of 40.8％ (71／174). The post-thaw survival rate of in vivo blastocysts (93.7％; 45／48) was significantly higher than that of in vitro blastocysts (82.5％; 52／63, P＜0.05). Embryo transfer was carried out using 8 Mongolian recipient cows and 2 post-thaw blastocysts per recipient. Five of 8 recipients were found pregnant at Day 60 but one abortion occurred by Day 240. Two of offspring were produced from the Mongolian cows at 275 days after embryo transfer. These results indicated that a GMP vitrification method could be used as a cryopreservation technique for in vivo or in vitro bovine blastocysts and produced effectively a Korean Native Cow following embryo transfer into a Mongolian recipient cow.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.239-244
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• 1998

The purpose of this study is compare IVF cycle outcome in poor responders between clomiphene citrate (CC) stimulated and controlled ovarian hyperstimulation (COH) protocol. A total of 94 patients responding poorly in previous IVF cycles (estradiol<600 pg/ml or less than 3 oocytes retrieved) subsequently underwent either COH (COH group: 122 cycles, 68 patients) or CC-stimulated cycles (CC group: 43 cycles, 26 patients). CC was administered for five consecutive days starting on cycle day 3 at a dose of 100 mg daily. Serial transvaginal ultrasound examination was done from cycle day 8. Urine was collected $3\sim4$ times before hCG injection for the detection of LH surge. The hCG was administered when serum estradiol reached greater than 150 pg/ml and mean follicle diameter>16 mm. In COH group, ovarian stimulation was done using short protocol (GnRH-a/FSH/HMG/hCG). No difference in age or number of transferred embryos was found between CC group and COH group. COH group had significantly (p<0.05) higher mean peak level of $E_2$ ($810{\pm}112$ vs $412{\pm}55$ pg/ml) and greater number of retrieved oocytes ($3.0{\pm}0.2$ vs $2.0{\pm}0.2$) than CC group. CC group had transferred embryos $(1.8{\pm}0.2)$ compared with $(2.1{\pm}0.2)$ in COH group. However, CC group had higher pregnancy rate than COH group per retrieval [26.9% (7/26) vs 6.2% (6/97)], or per transfer [31.8% (7/22) vs 7% (6/86)]. Although cycle cancellation rate in CC group (48.8%) was higher than that of COH group (21.3%), the pregnancy rate per cycle in CC group was still higher (16.3%) than COH group (4.9%). In addition, implantation rate in CC group was 17.5% (7/40), which was significantly (p<0.01) higher than 3.9% (7/180) in COH group. These data suggest that oocyte and embryo quality are lower in COH cycles of poor responders than CC cycles. We suggest that clomiphene citrate stimulated IVF cycle may be more efficient than COH IVF cycle in poor responders in terms of lower costs and higher pregnancy performance.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1403-1406
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• 2005

The present study was undertaken to study the effect of thermal stress on sexual behaviour of superovulated ewes. Fourteen adult Bharat Merino ewes with an average body weight of 29.4${\pm}$2.34 kg were randomly allocated into two groups of 7 each. All the animals were grazed on natural pasture in the morning and evening hours and housed in shed during night. Animals of Group-1 were housed in shed from 10:00 to 16:00 h while the animals of Group-2 were exposed to thermal stress in a hot chamber ($40^{\circ}C$ /6 h/day). All the animals were offered drinking water once a day at 16:30 h. Meteorological observations i.e. dry bulb, wet bulb, minimum and maximum temperature were recorded daily inside the shed as well as in hot chamber throughout the experimental period. For superovulation of animals, standard protocol developed at the Institute, using FSH (Ovagen 5.4 mg in eight injections) and PMSG (200 IU) was followed. Various sexual behaviour parameters (circling, tail fanning, head turning, standing and approaching to ram) and estrus incidence (onset of estrus and estrus duration) were observed in both the groups. The different estrus symptoms were graded subjectively on arbitrary scale of 0-5 where 0 representing no sexual behaviour (0%) and 5 representing maximum intensity in sexual behaviour (100%). Estrus was detected with the help of a marked aproned ram of proven vigor at six hourly intervals. The average percent values for sexual behaviour parameters recorded in Group-1 and Group-2 animals were 53.7${\pm}$3.76 vs. 41.1${\pm}$2.18 for circling, 71.8${\pm}$5.42 vs. 49.0${\pm}$4.41 for tail fanning, 64.7${\pm}$3.30 vs. 44.5${\pm}$4.34 for head turning, 90.1${\pm}$3.16 vs. 75.8${\pm}$4.02 for standing and 63.8${\pm}$4.8 vs. 41.9${\pm}$4.58 for approaching to ram. Animals exposed to thermal stress had significantly lower values of these sexual behaviour parameters. The animals kept in shed exhibited estrus earlier (25.4${\pm}$1.12 h) and duration was higher (37.7${\pm}$1.59 h) as compared to animals exposed to thermal stress i.e. 30.6${\pm}$1.16 h and 31.7${\pm}$3.57 h, respectively. The findings of the study indicate that thermal stress reduces the intensity of sexual behaviour in ewes and may result in failure of the animal to mate and conceive.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.21-26
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• 2007

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 $100{\mu}1$와 $10{\mu}1$ 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건($S;\;10{\mu}1$ 소적) 및 그룹 배양 조건 ($G;\;100{\mu}1$ 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 $1{\mu}g/ml$ $estradiol-17{\beta}$를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene glycol로 직접 이식법에 의한 동결 방법으로 동결을 실시하였고, 세포수는 융해 후 생존 수정란에 대해 조사하였다. 체외 배양 시간이 $16{\sim}17$시간 배양 조건에서 난자의 성숙율은 그룹 배양 조건$(27.1{\pm}16.8%)$보다는 단독 배양조건$(57.1{\pm}15.0%)$에서 성숙율이 높았다(p<0.05). 그러나 체외 배양 시간이 $18{\sim}19$ 시간과 $20{\sim}21$시간 배양시는 유사한 성숙율을 보였다. 난자의 체외 배양율은 체외 배양 시간의 증가에 의해 성숙도가 $86.3{\pm}9.9%$로 증가하였다. 접합체(zygote)의 분할율은 단독이나 그룹 배양 조건에서도 유사한 결과를 얻었다. 배반포 발달율은 배양 $7{\sim}8$일째에 조사한 결과 단독 배양 방법보다는 그룹 배양 방법에서 발달율이 높았으나, 분할된 접합체를 기준으로 한 경우 배반포 발달율$(S;\; 21.4{\pm}10.6%,\;G;\;39.0{\pm}13.1%)$에서는 유의적인 차이가 없었다. 단독 배양과 그룹 배양에서 $6.5{\pm}8$일 사이에 배반포로 발달된 수정란의 세포수 조사에서는 유사한 결과를 얻었다. 동결 융해 후 24시간 배양 후 배반포 생존율(5; 24.2%, G; 30.2%), 부화율(S: 20.9%, G: 12.7%) 및 생존 수정란수(S; 45.2%, G: 42.8%)에서도 배양 조건에 따른 유의적인 차는 없었다. 결론적으로 mSOF배양액을 이용하는 경우 미성숙 난자의 체외 성숙 유도 배양 시 단독이나 그룹 배양 시 배반포 발달율에서 그룹간에 유의적인 차가 인정되었다(p<0.01).

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.311-317
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• 2017

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.229-235
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• 2012

This research was investigated the relationship, in high-producing Holstein donor cows, between the number of the transferable embryos and the blood serum concentrations of Blood Urea Nitrogen (BUN), glucose and cholesterol, which affect the nutritional state of cows. CIDRs were inserted into the vaginas of twenty two heads of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with $PGF_{2{\alpha}}$ and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. The total numbers of ova collected from 3 experimental groups whose blood BUN concentrations were <10 mg/dl, 11~18 mg/dl and ${\geq}19$ mg/dl were 8.9, 12.5 and 19.0, respectively; whereas the numbers of transferable embryos were 5.8 + 1.9, 7.9 + 2.8 and 5.2 + 1.4, respectively. When glucose concentration was <60 mg/dl, the total number of collected ova was 9.9, which was smaller than when the concentration was 60~70 mg/dl or ${\geq}70$ mg/dl. When glucose concentration was 60~70 mg/dl, the number of transferable embryos was 7.1 + 2.4, which was slightly larger than the numbers 6.4 + 2.1 and 6.1 + 1.7 that were obtained when the concentrations were <60 mg/dl and ${\geq}70$ mg/dl, respectively ; however, the differences were not significant (p>0.05). When cholesterol concentrations were <150 mg/dl, 150~200 mg/dl and ${\geq}200$ mg/dl, the total numbers of collected ova were 11.2, 11.3 and 8.6, respectively. Whereas the numbers of transferable embryos were 7.1 + 2.1, 7.3 + 1.9 and 5.6 + 1.3, respectively ; however, the differences were again not significant (p>0.05). The result of this research showed no significant difference in ovum recovery rate and the number of transferable embryos according to major metabolite concentrations in high-producing Holstein donor cows. However, it is considered that the failure of maintaining proper nutritional status would cause the fall in in vivo embryo productivity.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.237-243
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• 2012

This research was investigated the relationship between the number of the transferable embryos and estrus expression rate, BCS (Body Condition Score), which affect the nutritional state of the cow, in Holstein donor cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with $PGF_{2{\alpha}}$ and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. When BCS was $$\leq_-$$2.5, the total number of collected ova was 7.3 + 1.9, which is significantly lower (p<0.05) than the numbers 15.4 + 2.8 and 15.4 + 2.1 that were obtained when BCSs were 2.75 and $$\geq_-$$3.0, respectively. Whereas the numbers of transferable embryos were 5.2 + 1.4 when BCS was $$\leq_-$$2.5, which was smaller than the numbers 6.0 + 2.1 and 8.5 + 1.8 that were obtained when BCSs were 2.75 and $$\geq_-$$3.0, respectively; however, the differences were not significant. As for estrus induction rate, the cow groups whose BCSs were 2.75 and $$\geq_-$$3.0 showed 100.0% and 95.0%, respectively. Whereas the cow group whose BCS was $$\leq_-$$2.5 showed 57.1%, and the differences were significant (p< 0.05). As for estrous expression rate, the cow groups whose BCSs were $$\leq_-$$2.5, 2.75 and $$\geq_-$$3.0 showed 100.0%, 100.0% and 85.7%, respectively; however, the differences were not significant. According to the result of this research, it is considered that the total number of collected ova and the number of transferable embryos will be affected by the nutritional state before and after in vivo embryo production and superovulation treatment, and that although the mechanism is not clear, poor stockbreeding management and nutritional level would cause the decrease of ovum recovery rate and the number of transferable embryos in high-producing cows. On the other hand, diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.