• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.300-304
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• 2019

This study was conducted to examine the oocyte recovery efficiency through having an OPU session once and twice a week. Also, the oocyte recovery efficiency was examined by using OPU after two and three months of rest period. Six cows were used for oocytes collection and were randomly divided into two groups. In experiment 1, OPU sessions were conducted once and twice a week to collect oocytes. The collected oocytes between once and twice OPU groups were classified into four groups (grade 1, 2, 3 and 4) according to the quality of cumulus cells and ooplasm. Based on the result, the percentage of collected oocytes per aspirated follicle number was similar between once and twice OPU session groups (65.5 ± 1.9 and 68.7 ± 1.4 vs.). However, the percentage of grade 1 oocytes from the twice OPU session group was significantly high compared with that of the once a week OPU session group (25.3 ± 0.9 and 32.5 ± 1.2% vs. once and twice session group, respectively, p < 0.05). In experiment 2, the group with three months of rest period tended to have a high percentage of collected oocyte compared with the group with two months of rest period (64.6 and 70.9% vs. 2 and 3 months rest group, respectively, p = 0.62). The percentage of grade 4 in the group with three months of rest period was significantly low compared with the group with two months of rest period group (27.3 and 36.5% vs. two and three months rest group, respectively, p = 0.05). In conclusion, twice a week OPU session is suitable for collection of high quality oocytes by using OPU, and three months of rest period is needed for the recovery of oocyte quality of a donor cow.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.50-56
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• 2019

These studies were conducted to evaluate developmental competence of follicular oocyte collected from the ovaries of Hanwoo cows with the high offspring meat quality (1++ and 1+ grade). Cumulus oocyte complexes from individual cows were matured, fertilized and cultured using protocols of in-vitro maturation (IVM), invitro fertilization (IVF) and in-vitro culture (IVC). The rates of blastocyst development from Hanwoo cows with the offspring meat quality grades of 1++ and 1+ were 18.6 and 21.2%, respectively. The rates of blastocyst development were 26.3, 20.7, 20.7, 17.2 and 31.2% from Hanwoo cows with the meat quality grades of 1++, 1+, 1, 2 and 3, respectively. Fiftyseven transferable embryos were recovered from 11 Hanwoo donor cows (5.2/head) with the high offspring meat quality grades of 1++ and 1+ in vivo, and the pregnancy rate after embryo transfer was 61.1%. In conclusion, these results suggest that in vitro embryo production from the ovaries of cows with the high meat quality grades using individual culture system can be used an efficient method for livestock improvement. In addition, for the successful industrialization of embryo transfer, conception rate should be improved.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.1-8
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• 2000

The ultrasound-guided oocytes cllection (ovum pick-up ; OPU) has become a substitution for superovlation in cattle. The objective of this study was to examine the effect of OPU frequency on the in vitro production of embryos in Hanwoo cattle. Six cycling Hanwoo cows were distributed into two groups for either once or twice weekly OPU sessions. Oocytes were collected by ultrasound-guided follicle aspiration(SA600) using a 6.5HMz transducer and attached with 18 gauge needle, with vacuum pressure of 40 mmHg. The cumulus-oocyte complexes (COCs) collected from each donor were matured in TCM 199 supplemented with 10% fetal bovine serum at 5% CO2 in air at 38.5$^{\circ}C$ for 22h and in vitro matured oocytes were co-incubated with sperm(separated by Percoll gradient) for 6h. The zygotes were co-cultured on cumulus cell monolayer in 10ul droplets in the same culture medium and conditions used for IVM for 7 days. On Day 7 of culture, development to blastocysts was examined. Although the number of oocytes collected was variable depending on individuals, overall embryo production in the twice per week OPU sessions was better that in the once per week sessions(6~21 vs 2~7 blastocysts produced, respectively). Two cows(E, A) were good oocyte donors and embryo production was superior in cow C ; however, cow F was a poor donor as compared to the others. In conclusion, these results suggest that for embryo production, twice weekly OPU sessions were better than once per week for producing embryos in vitro from Hanwoo cattle.

• Journal of The Korean Society of Grassland and Forage Science
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• v.38 no.4
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• pp.320-324
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• 2018

In this study, we examined effect of sperm penetration of oocytes after in vitro fertilization (IVF) with cauda epididymal spermatozoa in Hanwoo bull after feeding of timothy hay. One testicle with epididymides was castrated from one Hanwoo bull (14 months of age) and spermatozoa recovered from cauda epididymis and cryopreserved. As control, frozen Hanwoo semen was used. Matured cumulus oocyte complexes were co-incubated with frozen-thawed cauda epididymal spermatozoa for 12 or 18 hours. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid. In experiment 1, we examined sperm penetration rate at 12 hours of IVF with epididymal sperm. Total penetration rate among cauda epididymis and control was similar(mean${\pm}$standard error, cauda epididymis and control vs. $49.7{\pm}11.3$ and $54.4{\pm}12.8%$). In experiment 2, cleavage and blastocyst developmental rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar(cauda epididymis and control vs. $81.2{\pm}3.4$ and $82.7{\pm}2.5%$). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group(cauda epididymis and control vs. $24.4{\pm}1.6$ and $12.2{\pm}2.8%$, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high embryo developmental competence and can be used as an alternative to ejaculated frozen sperm in vitro.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.67-74
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• 1997

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5$^{\circ}C$ in 2% $CO_2$ in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2$0^{\circ}C$ water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100$\mu$M $\beta$-mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF$_2$$\alpha$(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

• Development and Reproduction
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• v.21 no.4
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• pp.407-415
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• 2017

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: $32.5{\pm}10.1%$ vs control: $77.8{\pm}5.3%$). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, $200{\mu}M$) and melatonin ($0.1{\mu}M$), in BIP/GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, $p-eIF2{\alpha}$, $eIF2{\alpha}$, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.305-310
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• 2019

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.39-45
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• 2009

This study was carried out to evaluate the nuclear, cytoplasmic maturation and developmental potential of bovine oocytes selected by brilliant cresyl blue (BCB) as indirect measurement of oocytes growth phase. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm follicles from slaughterhouse Hanwoo ovaries. The COCs were divided into stained cytoplasm to blue (BCB+) and unstained (BCB-) according to their ooplasm BCB coloration stained by $26{\mu}m$ of BCB after 90 min. Selected COCs were cultured in a TCM 199 for 18 to 26 h. Nuclear maturation and total cell number was evaluated after in vitro maturation (IVM) or in vitro culture (IVC) using $10{\mu}g/ml$ Hoechst 33342, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay before (0 h) and after (24 h) IVM. The oocyte diameters were not differed significantly between BCB+ ($157.4{\pm}5.8{\mu}m$) and BCB+ ($149.0{\pm}31.0{\mu}m$) groups (p>0.05). However, the proportion of metaphase II oocytes in BCB+ group was significantly higher than BCB- group after IVM (p<0.05). GSH content of BCB+ group oocytes was significantly higher than that of BCB- group just after collection ($7.3{\pm}0.6$ vs. $4.8{\pm}0.6\;pmol/oocyte$, p<0.05), but not varied after IVM($13.1{\pm}0.9$ and $12.6{\pm}2.5\;pmol/oocytes$ for BCB+ and BCB- respectively; p>0.05). The proportion of blastocyst formation and total cell number in BCB+ group (23.5% and $105.5{\pm}28.6$) was significantly higher than that in BCB- (9.8% and $72.4{\pm}26.1$; p<0.05). The results indicate that BCB+ group oocytes may provide a cellular and functional basis for the greater developmental competence in Korean Native Cow (KNC) oocytes.

• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.