Optimization of In Vivo Embryo Production and Pregnancy following Embryo Transfer in Hanwoo Cattle

Jeon, Soon-Hong,Jung, Kyoung Sub,Choi, Jae-Won,Heo, Young-Tae,Xu, Yong-Nan,Kim, Nam-Hyung The Korean Society of Embryo Transfer 2013 한국동물생명공학회지 Vol.28 No.4

Embryos formed in vivo were collected from 171 donors housed in Chung Cheong Buk-Do Institute of Livestock and Veterinary Research of the Chungbuk community during the years 2009~2012. We evaluated annual embryo collection, effect of follicle stimulating hormone (FSH), controlled internal drug release (CIDR) and prostaglandin (PG) administration to the donor for superovulation and controlling the estrus cycle, seasonal effects of embryo collection and compared the number of embryos recovered as per the collection days and pregnancy rate. In all, 1,243 embryos were collected from 118 donors with an average of $7.31{\pm}5.35$ embryos per donor, out of which 69.4% were transferable. Dosages of FSH required for inducing superovulation in various donors were compared. Average number of embryos collected from donors administered with 30 AU of FSH ($7.13{\pm}5.74$ per donor) was not significantly different from that of donors who were given an injection of 24 AU of FSH ($7.53{\pm}4.91$ per donor). However, the percentage of transferable embryos in the 30AU FSH-administered group (63.2 %, 449 of 711) was higher than that in the 24AU FSH-administered group (77.8%, 414 of 532). In the group of donors under a natural estrus cycle, the FSH dose administered did not influence the number of transferable embryos produced ($7.49{\pm}6.25$ per donor for 30 AU of FSH vs $7.49{\pm}4.92$ per donor for 24 AU of FSH). However, in donors administered with CIDR and PG for controlling the estrus cycle, the FSH dose affected the average number of transferable embryos collected ($4.25{\pm}2.87$ per donor for 30 AU of FSH vs $8.50{\pm}6.36$ per donor for 24 AU of FSH). We collected embryos from donors 6, 7 or 8 days after artificial insemination (AI). Results showed that the percentage of transferable embryos among those collected 8 days after AI was significantly higher than that among embryos collected 6 or 7 days after AI. Seasonal variations did not affect number of recovered embryos and pregnancy rates in natural estrus cycle and CIDR treatment groups (48.28% and 42.55%) but higher than pregnancy rate of frozen embryos (19.63%). These results indicated that administration of FSH beyond a threshold dose (at least 24 AU) has no beneficial effect on the production embryos and that collection of embryos 7~8 days after AI is optimal for embryo recovery. CIDR treatment induced superovulation in short term and had no influence on the natural estrus cycle. Finally, although good-quality embryos were transferred, freezing significantly reduced the pregnancy rates after transfer.

Embryo Collection, Transfer and Pregnancy of Riding Horses : First Successful Case in Korea

Park, Yong-Soo,Yang, Jae-Hyuk,Cho, Young-Jae,Oh, Dong-Yep,Cho, Gil-Jae The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.2

Embryo transfer (ET) could be a relevant tool for genetic improvement programs in horses similar to those already underway in other species and produce multiple foals from the same mare in one breeding season. However, there have been no reports describing equine embryo transfer performed in Korea. In the present study, we performed an equine embryo collection and transfer procedure for the first time. We examined the embryo collection and pregnancy, size of embryo during the incubation period after collection, and progesterone (P4) and estradiol-$17{\beta}$ (E2) concentrations in mare's serum at embryo collection and transfer. A total of 16 donors responded to estrus synchronization; estrus was induced in 12 donors and 4 recipients, and artificial insemination was successful in 10 donors and six blastocysts were collected from donors. Of these blastocysts, we monitored the size of blastocysts for 3 day during incubation and transferred 2 blastocysts to a recipient, with 1 successful pregnancy and foal achieved. The dimensions of equine embryo at day 7 to day 9 were $409{\mu}m$, $814{\mu}m$ and $1,200{\mu}m$. The serum P4 and E2 concentrations were $7.91{\pm}0.37ng/{\mu}L$ and $45.45{\pm}12.65ng/{\mu}L$ in the donor mare, and 1$6.06{\pm}3.27ng/{\mu}L$ and $49.13{\pm}10.09ng/{\mu}L$ in the recipient mare.

Laparoscopic Transabdominal Transfer of Blastocysts in Korean Black Goats

Cho, Sang-Cheol,Cho, Jong-Ki,Shin, Sang Tae The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.2

As a part of the effort to improve post-transfer survival rate of embryos in Korean black goats, a technique for laparoscopic uterine transfer of blastocysts was carried out. A total of 26 transferrable embryos (morula to expanded blastocysts) were transferred to 13 recipient goats via transabdominal laparoscopic method. In consequence of our hormone protocol, 65% of the recipients (13/20) were found to have synchronized estrus. After confirmation of corpus luteum in each recipient goat, a Babcock laparoscopic forceps was inserted into the lower abdominal cavity to hold a uterine horn and fasten it near the peritoneum without causing injury. Then 7.5cm long 16G IV catheter was inserted directly into the uterine lumen through the abdominal wall. After removal of the stylet of the IV catheter, the embryo transfer tube (identical in size to the stylet and loaded with blastocysts) was inserted into the uterine lumen through the catheter to unload the embryos. Of the 13 estrus synchronized recipients, 9 were transferred blastocysts and 4 were transferred molurae (2 embryos in each recipient) in uterine ipsilateral to the ovary with corpus luteum. Four of the 9 recipients which blastocysts were transferred using this method has been confirmed pregnant (44.4% pregnancy rate).

Influence of Oxygen Consumption on Pregnancy Rates of Hanwoo Calves following Embryo Transfer

Kim, Hyun,Bok, Nan-Hee,Kim, Sung-Woo,Do, Yoon-Jung,Seong, Hwan-Hoo,Kim, Jae-Hwan,Kim, Dong Hun,Kim, Min-Kyu,Ko, Yeoung-Gyu The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.3

Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we investigated the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of apoptosis-related genes was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts ($10.2{\times}10^{14}/mol\;s^{-1}$ versus $6.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0 and over $12.0{\times}10^{14}/mol\;s^{-1}$, respectively. Apoptosis regulatory genes, Hsp-70.1 were significantly increased in over-10.0 group than below 10.0 group but in Caspase-3, Bax and P53 gene, there was no significant difference. In conclusion, These results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

Comparisons of Development Potential in Bovine SCNT Embryos using Donor Cells treated with Different Demethylating Inhibitors

Jeon, Byeong-Gyun,Jeong, Gie-Joon,Rho, Gyu-Jin The Korean Society of Embryo Transfer 2015 한국동물생명공학회지 Vol.30 No.3

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p<0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

Effect of uterine histotroph on embryo development in pigs

Han, Hye-In,Lee, Sang-Hee,Song, Eun-Ji,Lee, Seunghyung,Cheong, Hee-Tae,Yang, Boo-Keun,Park, Choon-Keun The Korean Society of Embryo Transfer 2016 한국동물생명공학회지 Vol.31 No.3

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.

Effect of L-Glutathione Treatment during Somatic Cell Nuclear Transfer Procedures on the Subsequent Embryonic Development and DNA Methylation Status of Cloned Bovine Embryos

Bae, Hyo-Kyung,Yoon, Nam-Sik,Hwang, In-Sun,Park, Choon-Keun,Yang, Boo-Keun,Cheong, Hee-Tae The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.4

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with $50{\mu}M$ GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group ($32.5{\pm}1.2%$, 78/235) than that of non-treated control SCNT embryos ($22.3{\pm}1.8%$, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group ($2.3{\pm}0.4%$) were significantly lower (P<0.05) than that of control ($3.8{\pm}0.6%$). Relative caspase-3 activity of GSH treated group was $0.8{\pm}0.06$ fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group ($13.1{\pm}0.5$, pixels/embryo) was significantly lower (P<0.05) than that of control ($17.4{\pm}0.9$, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

Optimization of Post-Activation Systems to Improve the Embryonic Development in Porcine Parthenogenesis and Somatic Cell Nuclear Transfer

Roy, Pantu Kumar,Kim, Ghangyong,Fang, Xun,Hassan, Bahia MS,Soysa, Mahanama De,Shin, Sang Tae,Cho, Jong Ki The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.3

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

Study on Chemicals for Post-activation in Porcine Somatic Cell Nuclear Transfer

Min, Kyuhong,Na, Seungwon,Lee, Euncheol,Kim, Ghangyong,Yu, Youngkwang,Roy, Pantu Kumar,Fang, Xun,Salih, MB,Cho, Jongki The Korean Society of Embryo Transfer 2016 한국동물생명공학회지 Vol.31 No.2

Since the first success of animal cloning, somatic cell nuclear transfer presented various ideas in many research areas such as regenerative medicine. However, SCNT embryos has poor survival rate. Therefore, numerous researches carried out to enhance the developmental capability of porcine nuclear transfer embryos. Cytochalasin B, demecolcine, latrunculin A, cycloheximide and 6-dimethylaminopurine are efficient chemicals treated in post-activation procedure to increase the efficiency of SCNT. This review study is aim to investigate the effects of these chemicals applied to post-activation in porcine SCNT. Cytochalasin B, demecolcine, latrunculin A are cytoskeletal manuplators inhibit extrusion of pseudo-polar body. Cytochalasin B and demecolcine showed considerably higher blastocyst formation proportion (26-28%) compared to when they are not treated (16%). And when latrunculin A was treated for postactivation, blastocyst formation proportion was increased in SCNT embryos exposed to LA (38%) than those in control (14%). On the other hand, cycloheximide and 6-dimethylaminopurine are protein synthesis and kinase inhibitors. And they help to maintain $Ca^{2+}$ fluctuation in oocytes. Cleavage and blastocyst rates of NT embryos were increased when they were exposed to CHX (16.9% and 5.4% with no CHX).And 6-DMAP also showed higher blastocyst formation (21.5% compared to 15.7%, control). Although all these chemicals have different mechanisms, they showed developmental competence enhancement in NT embryos. However, there are only few studies comparing each chemical's post-activation effect. Therefore, further research and study should be conducted to find optimal chemical for improving the efficiency of SCNT.

Establishment of Efficient Microinjection System in the Porcine Embryos

Malaweera, Don Buddika Oshadi,Ramachandra, Sisitha,Wu, Jun-Bo,Oh, Seung-Kyu,Kim, Seung-Hwan,Kim, Seok-Joong,Shin, Sang-Tae,Cho, Jong-Ki The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.1

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.