Investigation of Feline Ovulation Time after LH Surge Induced by hCG Injection in Superovulation

전교희,김성환,이진구,김광용,오성규,Don Buddika Oshadi Malaweera1,Sisitha Ramachandra,윤기영,신상태,조종기 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.2

Feline ovulation time after LH surge have not been defined because its LH surge is occurred by several times ofcoital vaginal induction and cat has relatively longer time between LH surge and ovulation compared with othermammalian species. This study was performed to investigate the feline ovulation time after LH surge that was inducedby hCG injection for superovulation with PMSG. For superovulation, all cats were received an initial injection ofPMSG (200 IU, i.m.) followed 80 hrs later with an injection of hCG (200 IU, i.m.). And then, sampling of bothovaries was surgically performed at each 6 different times (10, 18, 22, 26, 29, and 32 hrs) after hCG injection. Cumulus-oocyte-complexes (COCs) were collected from 2 sides of oviducts and ovaries were fixed for ovarianhistology. Total 38 COCs were collected only at hCG 32 hrs and no COCs were shown at earlier 5 times. However,in the ovarian histology, corpus haemorrhagicum or corpus luteum was not shown in all groups including ovary athCG 32 hrs that COCs were collected. In conclusion, it was suggested that feline ovulation was occurred at 29∼32hrs after LH surge and taken relatively long time for CL formation after ovulation.

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.

Establishment of Efficient System in the Microinjection of TALEN mRNA into Porcine Embryos

Don Buddika Oshadi Malaweera,Sisitha Ramachandra,Junbo Wu,Seungkyu Oh,Seunghwan Kim,Seokjoong Kim,Goo Jang,Jongki Cho 한국동물생명공학회(구 한국동물번식학회) 2013 발생공학 국제심포지엄 및 학술대회 Vol.2013 No.1

To investigate the efficient laboratory techniques for the injection of TALENs mRNA into porcine parthenogenesis and IVF embryos we had explores the injection time, volume and concentration, for the efficient blastocyst production has been identified within the experimental period. In experiment 1, to investigate injection time, compared 4 different time durations (1 hr, 2 hrs, 4 hrs & 5 hrs) after post activation of parthenogenetic embryos. There is no significant difference between four groups regarding the percentage of cleavages (55%). But comparatively higher percentage (20%) of blastocyst can be observed in the groups of 1hr and 2hrs injection after post activation. Even though observed higher blastocysts, only EGFP expressed blastocysts (2.2%) taken from the treatment of injection after 4 hrs of post activation. In experiment 2, compared two different concentration of EGFP injection in to embryos. 20 ng/μL and 50 ng/μL two concentrations were injected to the parthenogenetic embryos after 4 hrs of post activation and observed blastocysts after 7 days of incubation. There were significant difference between two treatments of concentrations considering percentages of cleavages and blastocysts. 50 ng/μL concentration injected embryos capable enough to cleaved up to 39% and produces blastocysts 10%, meanwhile 20 ng/ μL concentration illustrates 62% cleavage and 16% blastocysts percentages. Only 20 ng/μL injected embryos capable enough to express EGFP (3.1%) after 7 days of incubation. These results implies that 20 ng/μL concentration and injection 4hrs after post activation more effective injection conditions for microinjection.

Establishment of Efficient Microinjection System in the Porcine Embryos

Don Buddika Oshadi Malaweera,Sisitha Ramachandra,Jun-Bo Wu,Seung-Kyu Oh,Seung-Hwan Kim,Seok-Joong Kim,Sang-Tae Shin,Jong-Ki Cho 韓國受精卵移植學會 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 (20 ng/μl and 50 ng/μl) 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 20 ng/μl than the 50 ng/μl in parthenogenetic embryos. In IVF embryos, only 20 ng/μl 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 20 ng/μl concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2∼4 hrs after IVF, 20 ng/μl concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

Effect of 7,8-Dihydroxyflavone on In Vitro Maturation of Oocytes in Pigs

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

In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, $2{\mu}m$) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the $1.0{\mu}m$ group compared with another 4 groups ($83.3{\pm}2.1$ vs. $80.7{\pm}1.4$, $79.8{\pm}1.4$, $78.3{\pm}1.2$, $79.4{\pm}1.6$), respectively (P<0.05). In the embryo culture, $1.0{\mu}m$ group also showed the significant higher cleavage rates ($76.8{\pm}3.1$ vs. $62.1{\pm}5.0$, $65.7{\pm}4.0$, $68.6{\pm}3.7$, $64.6{\pm}4.0%$) and blastocyst formation rates - ($39.6{\pm}4.0%$ vs. $28.6{\pm}3.3$, $31.1{\pm}3.9$, $29.3{\pm}2.5$, $39.6{\pm}4.0$, $26.4{\pm}3.2%$), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of $1.0{\mu}m$ of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

Development of in vitro produced porcine embryos according to serum types as macromolecule

손정민,Don Buddika Oshadi Malaweera,이은송,신상태,조종기 대한수의학회 2013 JOURNAL OF VETERINARY SCIENCE Vol.14 No.3

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum,prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.

Effect of 7,8-Dihydroxyflavone on In Vitro Maturation of Oocytes in Pigs

Seung-Kyu Oh,Don Buddika Oshadi Malaweera,Sisitha Ramachandra,Sang-Tae Shin,Jong-Ki Cho 韓國受精卵移植學會 2014 한국동물생명공학회지 Vol.29 No.1

In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, 2 uM) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the 1.0 uM group compared with another 4 groups (83.3 ± 2.1 vs. 80.7 ± 1.4, 79.8 ± 1.4, 78.3 ± 1.2, 79.4 ± 1.6), respectively (P<0.05). In the embryo culture, 1.0 uM group also showed the significant higher cleavage rates (76.8 ± 3.1 vs. 62.1 ± 5.0, 65.7 ± 4.0, 68.6 ± 3.7, 64.6 ± 4.0%) and blastocyst formation rates - (39.6 ± 4.0% vs. 28.6 ± 3.3, 31.1 ± 3.9, 29.3 ± 2.5, 39.6 ± 4.0, 26.4 ± 3.2%), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of 1.0 uM of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

Improved Development Competence using Immortalized Cells as Donor in Bovine Nuclear Transfer

Sisitha Ramachandra,Don Buddika Oshadi Malaweera,Junbo Wu,Seungkyu Oh,Seunghwan Kim,Woojae Choi,Goo Jang,Jongki Cho 한국동물생명공학회(구 한국동물번식학회) 2013 발생공학 국제심포지엄 및 학술대회 Vol.2013 No.1

Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells.

Choi, WooJae,Kim, Eunji,Yum, Soo-Young,Lee, ChoongIl,Lee, JiHyun,Moon, JoonHo,Ramachandra, Sisitha,Malaweera, Buddika Oshadi,Cho, JongKi,Kim, Jin-Soo,Kim, SeokJoong,Jang, Goo Landes Bioscience 2015 Prion Vol.9 No.4

<P>Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180??days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle.</P>

Investigation of Feline Ovulation Time after LH Surge Induced by hCG Injection in Superovulation

Jeon, Kyo-Hee,Kim, Seung-Hwan,Lee, Jin-Goo,Kim, Ghang-Yong,Oh, Seung-Kyu,Malaweera, Don Buddika Oshadi,Ramachandra, Sisitha,Yoon, Ki-Young,Shin, Sang-Tae,Cho, Jong-Ki 韓國受精卵移植學會 2014 한국동물생명공학회지 Vol.29 No.2

Feline ovulation time after LH surge have not been defined because its LH surge is occurred by several times of coital vaginal induction and cat has relatively longer time between LH surge and ovulation compared with other mammalian species. This study was performed to investigate the feline ovulation time after LH surge that was induced by hCG injection for superovulation with PMSG. For superovulation, all cats were received an initial injection of PMSG (200 IU, i.m.) followed 80 hrs later with an injection of hCG (200 IU, i.m.). And then, sampling of both ovaries was surgically performed at each 6 different times (10, 18, 22, 26, 29, and 32 hrs) after hCG injection. Cumulus-oocyte-complexes (COCs) were collected from 2 sides of oviducts and ovaries were fixed for ovarian histology. Total 38 COCs were collected only at hCG 32 hrs and no COCs were shown at earlier 5 times. However, in the ovarian histology, corpus haemorrhagicum or corpus luteum was not shown in all groups including ovary at hCG 32 hrs that COCs were collected. In conclusion, it was suggested that feline ovulation was occurred at 29~32 hrs after LH surge and taken relatively long time for CL formation after ovulation.