The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to 0.1 μM SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and 10 μM during the first 22 h of IVM to determine a suitable concentration, 0.1 μM SNAP (54.2%) exhibited a higher blastocyst formation than 0 and 10 μM SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG (45.3 ± 6.2%) compared to control (25.6 ± 3.4%). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG (89.3 ± 1.9%) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG (94.6 ± 1.1%). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation (27.8 ± 4.9%) compared to no treatment (41.4 ± 1.9%) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM (37.4 ± 3.4% and 34.4 ± 2.6%, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

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 μg/mL), CB (7.5 μg/mL) + CHX (10 μg/mL), CB (7.5 μg/mL) +DC (0.4 μg/mL), and CB (7.5 μg/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.

Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. Overall of the current studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. The purpose of this study is the effects of GDF8 on porcine parthenogenesis (PA) embryo development during in vitro culture (IVC). We were investigated the effect of GDF8 supplement during PA embryo IVC by cleavage and blastocyst formation rate and patterning analysis. Data were analyzed by on way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC followed experiment design as control, 0.2, 2, and 20 GDF8 supplement groups. After 48h of embryo culture time, no significant difference was observed on cleavage rate from the different concentration (0, 0.2, 2, and 20 ng/ml) of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After 120h of embryo culture time, the 0.2 and 2 group showed significantly (p<0.05) higher blastocyst formation rate than control (40.4% and 36.4% VS 40.4%, respectively). In embryo developmental pattern analysis, the 0.2 ng/ml GDF8 supplement groups showed significantly higher (p<0.05) 2-3 cell cleavage- and early blastocyst pattern compared with control (12.0% and 10.4% VS 6.6% and 6.2%, respectively). However there are no significantly different pattern was observed in other groups. In conclusion, the 0.2 ng/ml of GDF8 supplementation during porcine PA embryo IVC significantly changed embryonic developmental patterns. However there are further studies are required such as analysis of blastocyst total number, specific gene transcription pattern, and ICM/TE rate to make clarify and support the conclusion.

CRISPRs(clustered regularly interspaced short palindromic repeats) / CRISPR - associated(CAS) system has been used genome editing technology. Genome stage modification using CRISPR/CAS9 system can be used to wide research for the gene functional study and therapeutics. However, improving of CRISPR/CAS9 system in efficiency is essential for application in various fields. Here, we treated various chemicals during the procine early embryo development to increase the mutation of target site by NHEJ(non-homologous end joining). Firstly, we confirmed the chemical toxicity after parthenogenetic activation and then check embryo puality using by counting of total cell number and TUNEL Assay in blastocyst satge. To check any improvement on mutation rate by NHEJ pathway. AZT(3′-Azido-3′-deoxythymidine, antiretroviral drug – 0.1 μM) was treated after injection of cas9 and sgRNA target to OCT4 exon 5 during the zygote stage, followed by PCR sequencing. As a result, AZT treated group shows a significantly increased in knock-out efficiency as a consequence of NHEJ. Nocodazole(anti-neoplastic agent – 200ng/ml), RO-3306 (specific inhibitor of CDK1 - 10 μM) and NU-7026(PKC signalling inhibitor - 50 μM) was treated after injection of cas9 and sgRNA with eGFP vector during the zygote stage(hpa8~hpa20) and checked a efficiency of knock-in by PCR sequencing. Interestingly, nocodazole treatment groups increased of insertion of eGFP sequence in blastocyst stage compared with non-treat group(control : 8.33%, nocodazole treatment : 16.67%). However, RO-3306 and NU-7026 made a no impact. In summary, CRISPR/CAS9 system with treatment of chemicals during porcine embryogenesis can be improving of site-specific mutation and enhancement of CRISPR genome editing.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitromatured pig oocytes and treated for 4 h after electric activation with 0.5 μM latrunculin A (LA), 10.4 μM cytochalasins B (CB), and 4.9 μM cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8～88.6%), blastocyst formation (30.7～ 32.4%), and mean cell number of blastocysts (33.5～33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5～78.0%) and mean blastocyst cell number (33.6～35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5～85.7%) and blastocyst cell number (41.1～43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes () was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage () and mean number of cells in blastocyst ( cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 FSH,i numectivelo. In SCNT, fusion () of cell-cytoplast couplets and siosequent embryo cleavage () were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 FSHr(25% vs. ). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 7782%) and embryo cleavage (75% vs. 8690%) after SCNT than those matured in other media but blastocyst formation was not altered (1327%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

For many years, experience has been accumulated on embryo and gamete manipulation in mammals, The present work is an introduction of these techniques and their possible application in human embryology in s pecific cases, Mammalian c1on ing has been studied by many groups, but the success rate is sti ll low‘ Removal of maternal chromosomes from unfertil ized oocytes and injection of donor cells into enucleated oocytes are the most important factors for the improvement of cloning effi cien cy, Here, we introduce a novel one-step rnicromanipulation (OSM) system and laser-assisted zona pellucida piel'cing technique (LAZP) , 1n genera l, somatic cell nuclear transfer (SCNT) is completed by many processes including enucleation and donor cell fusion , Howevel', OSM is a simple method because donor cell is directly injected into ooplasm without fusion pl'ocess, 1n addition, chromosomal enucleation and donor cell inj ec tion are perfOl‘med simultaneously in OSM, While OSM was a pplied to porcine SCNT, LAZP was a pplied to murine SCNT, This rninirni zed the use of piezo-dri ven micromanipul ator (P1EZO) , I'educing chances 0 1' problems caused by P1EZO pulses, LAZP reduced time that took to pierce zona pellucida in removal of nucleus fl'om oocyte and somatic cell injection, which might have taken longer time with P1EZO, The simple , new OSM and LAZP system may help to enable large scale cloning by reduction of procedural steps, Pa l'thenogenesis de scribes the growth and development of an embryo without fertilization by a male Parthenogenetic ES cell s (PESCs) can be a useful cell source for tissue I'epail‘ and I'egeneration , Moreover , the defects in full-term developrnent of this PESCs enable researc hers to avoid the ethical concern , Here, the author showed that PESCs can differentiate into osteogenic lineage, The PESCs were induced osteogenic dlfferentlatlon The osteoblas t-specific gene expression such as osteocalcine, osteopontine, osteonectin, bone-sialo protein‘ coll agen type-l and alka line phos phatase showed osteogenic potential of differentiated PESCs, The author also focused on the neuronal induction of murine PESCs by simplified neurona l induction system to generate doparninergic (DA) neurons , As a result , PESCs were differentiated into nestin and Tuj-l positive cell s successfully, a lthough t he generation of DA neuron was Illruted For murine embryo cul ture, novel oil-free microtube cul tu re system was applied , This new culture system provides oil-free cu ltu re condi t ions and is easy to handle It was also associated with faster development and mOl'e t l'ophectodel'mal cells , which will enhance the development of murine embl'Yos to fur t hel' stages ,