• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.115-121
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• 2014

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, $0.1{\mu}g/mL$, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by $0.01{\mu}g/mL$ compared with other groups (p< 0.05). Moreover, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.89-95
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• 2000

This study was to establish an effective dilution technique in a vitrification of bovine blastocysts for the field trial. For vitrification, blastocysts were exposed in glycerol (G) and ethylene glycol (EG) mixture in m-DPBS supplemented with 10% FBS. Blastocysts were first exposed to 10% (v/v) G for 5 min, and subsequently were transferred to 10% G plus 20% EG (v/v) for 5 min. Finally, embryos were transferred to 25% G plus 25% EG (v/v) for 30 see and were placed in nitrogen vapor for 3 min, and then were plunged into L$N_2$. At thawing, the straw containing blastocysts was placed in air for 10 sec, and then plunged into a water bath at $25^{\circ}C$ until all ice had disappeared. They were placed in $25^{\circ}C$ and 36$^{\circ}C$ water according to treatment group for different time. Also, in vitro survival was assessed by the re-expansion and hatched rates at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) In the survival rates of vitrified bovine blastocysts according to different dilution time at thawing, the data of 1 min group (86.6, 56.6%) were higher than those of other treatment groups (2 min; 93.5, 35.4%, 2.5 min; 76.9, 30.7%, 3 min; 88.8, 36.1% and 3.5 min; 83.7, 8.1%). 2) When the in vitro survival of vitrified groups according to different developmental stage was examined at 48 h after thawing using 1 min dilution method, the hatching rates of fast developed embryos (expanded blastocyst: 81.3%: early hatching blastocyst: 86.2%) were higher than that of delayed developed one (early blastocyst: 46.6%). 3) In addition, when the in vitro survival of vitrified groups according to different embryo age was compared, the hatched rates at 48 h after thawing of Day 7 (66.6%) and Day 8 embryos (60.0%) were significantly higher than that of Day 9 embryos (22.7%) (P＜0.05). These results demonstrate that vitrified bovine IVM/IVF/IVC blastocysts can be successfully survived in vitro using one-step dilution (1 min) method.

• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.11.1-11.6
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• 2016

Background: This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods: The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results: The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9, and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 +10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %). Conclusions: Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.257-263
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• 1995

소 체외수정란의 체외배양 체계는 현재 완전히 확립되지는 않은 상태로서 8∼16 세포기 발육억제현상, 수정란의 파편하 현상, 성장지연 등 여러 가지 문제점들이 현 배양체계에서 나타난다. 그러나 hepler cell들과의 공배양에 의해 이러한 문제점들은 상당히 극복되며 또한 체외발생의 촉진 및 공배양된 수정란의 이식에 의해 임신율도 향상된다. 현재 소 체외수정란의 공배양에는 난관상피세포가 가장 널리 이용되나 이러한 시스템은 몇가지 문제점이 있다. 즉, primary culture를 확보하기 위하여 신선한 조직을 주기적으로 채취해야 하며 따라서 난관채취에 시간이 소요되고, 불편하며 또한 공배양에 이용되는 세포들이 균일하지 않은바 난관상피세포에 따라 배 발생 촉진작용에 변이를 나타내기도 한다. 그러나 확립된 cell line을 이용할 수 있다면 이러한 난관상피세포의 이용에서 나타나는 문제점들이 해결될 수 있다. Buffalo Rat Liver cell은 이러한 목적에 이용될 수 있는 cell line 중의 하나로서 이들은 여러 가지 성장인자(growth factor)를 분비하는 것으로 알려져 있다. 따라서 본고에서는 소 체외수정란의 공배양을 위한 BRL(Buffalo Rat Liver)cell의 이용성, 이용방법 및 이용에 있어서 고려하여야 할 요인들에 대하여 살펴보고자 한다.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.211-218
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• 2002

These studies were carried out to investigate the effects of the addition of amino acids and FBS as source of exogenous nitrogen fixation added to medium on in vitro production of blastocyst derived from bovine follicular oocytes. The base medium was TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and YS solution for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1) after IVF. Rmbryos were cultured in drop-culture that contained 25 embryos per 10 ${mu}ell$. The results obtained are as follows: 1 The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with NEAA derived from MEM alone were higher than those of YS solution without NEAA. 2. The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with EAA derived RPMI 1640 alone were significantly higher than those of YS solution without EAA (p<0.05). 3. When added to EAA on day 5 after NEAA supplementation on day 1, the developmental rates of hatched blastocyst and blastocyst to hatched blastocyst were improved. 4. When removed to EAA on day 3, day 4 and day 5 from medium after NEAA and EAA supplementation on day 1. the developmental rates of blastocyst to hatched blastocyst were reduced. 5. When added to FBS as source of exogenous nitrogen fixation, the developmental rates of blastocyst and hatched blastocyst that developed from the later culture higher(day 5) than those of the early culture.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.169-176
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• 2005

Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.169-175
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• 2013

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of $100{\mu}g$ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.