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), in-vitro 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.

The purpose of this study was to analyze whether FSH and LH hormone treatment directly or indirectly affect embryo development in embryonic development. To determine this, we compared the development of embryonic cells through the expression pattern of MMPs. As a result, 33.8% of blastocysts were formed in FSH added group, 20.8% in LH added group and 10% in FSH + LH added group. In addition, the activity of MMP-9 was highly detected in the FSH-added group, and the expression of Casp-3 was much lower than that of the other groups. These results suggest that the addition of FSH seems to increase the activity of MMP-9 in embryonic cells, and that LH, on the contrary, may activate MMP-2 activity. In addition, the expression level of MMP-2 in the FSH-added group was high in the Trophoblast cell group and in the LH-added group, the hormone ideal secretion might affect the development of the embryonic cell.

Up-to-date artificial insemination (AI) using frozen sperm consider as the most widely using technology for improvement of Korean Native Cow (Hanwoo) embryo production. However, it is time consuming, required at least 15~20 years to make more than 6 generations, and their offspring number is limited. To overcome such limitations, superovulation and in vitro fertilization have been developed. For superovulation, the number of produced embryos are not enough for commercialization and donor cows need rest period. This led to use of slaughterhouse ovary for in vitro fertilization, but it is impossible to repeat the collection from the same individual and it only can improve the genetic merits of offspring for one generation. Production of embryos using Ovum Pick-Up (OPU) technique, where oocytes can be repeatedly collected from living elite donor, might overcome these limitations. In this study, we investigated the possibility of using OPU technique from donors at different age and different session periods for mass-embryo-production. Oocytes were collected from 26 donor cows twice per week, 3 - 4 months per year, between 2013 and 2016. Results showed that, the average number of embryo produced in first year used donor was significantly higher than that in second year used donor (3.89 ± 2.85 vs 3.29 ± 2.70), however, there was no significant difference between third year used donor (3.51 ± 3.32) and other groups. Taken together, our data showed that repeated using of donor up to three years is possible for in vitro embryo mass-production. Moreover, OPU can be used as suitable embryo producing technique for livestock breed improvement.

The ovum pick up(OPU) technique can be used to produce embryos after in vitro culture of ovarian oocytes, can be used for early securement for effective herd early proliferation and excellent Hanwoo genetic resources, It is attracting attention as a very important technique for establishing technology. In addition to in vitro culture techniques, the number of oocytes retrieved depends on the environment and timing of the OPU. This study was conducted to compare and examine seasonal effect to the differences in the number of recovered oocytes, recovery rate and embryo development rate using Korean cattle kept in animal genetic resource research center by OPU technique. The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The recovery rate was 47.8±3.4% in summer (June to August) and 51.6±3.8% in autumn (September to October). The number of oocytes was 5.7±0.6 in summer and 5.2±0.7 in autumn. Oocyte grade A and B was 46.2%±6.3% in summer and 51.1±5.0% in autumn. The cleavage rate was 46.1±7.1% in summer and 65.0±11.3% in autumn. Blastocyst development rate was 19.9±9.4% in summer and 29.0±8.7% in autumn. There was no difference the recovery rate of oocytes and the number of embryos between summer and autumn. Cleavage rate and blastocyst rate of autumn was higher than summer. we will investigate to study the appropriate method for the production of Hanwoo embryos and the systematic comparison.

Sex preselection has always generated great interest among livestock producers due to an increase in the profitability of the cattle industry through the production of offspring with desired sex, such as females for dairy or males for meat production. Among the prevalent sorting methods, the embryo developmental potential is still very low as expected, and there is distinguished evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing lifespan. The consequence is a very low embryo development rate using sex-sorted semen, and its negative impact influences the progress of the dairy industry. Here, we established a new approach with reduced stress by using WholeMom® and observed no significant differences (P < 0.05) in early cleaving embryos between sorted X sperm and the control group, although there was a remarkable significant difference in embryos of the Y sperm group, 81.82 ± 2.71% vs. 87.44 ± 3.02% vs. 54.21 ± 2.21%, respectively. The percentage of embryos that developed into blastocysts (Day 7) was also significantly (P < 0.05) higher in the control and X Sperm group compared to the Y sperm group, 35.53 ± 1.92% and 29.76 ± 2.38% vs. 21.90 ± 1.54%. Moreover, B-SRY F2 and B-SRY R2 gene expression data exhibited 81.03% accuracy for the female embryos and 72.54% for the male embryos produced in vitro. And also the field trials for the heifer production using WholeMom by Artificial Insemination technique demonstrated 76% female and 24% male in vivo. In conclusion, the combination of pre-selected sex semen and OPU derived elite cattle embryo production is highly recommended to apply to the mass production in the dairy industry with rapid genetic up-gradation.

Thiamethoxam (TMX) is a neonicotinoid insecticide. Residues of TMX have been detected in various crops. Although it has specific high toxicity to insects and is designed to exterminate them, the toxicity has also found in mammals recently. Differ from acetylcholine toxicity, TMX has peroxide toxicity in mammals. Matured oocytes have the capacity of fertilization, but oocytes own abundant mitochondria and its maturation is vulnerable to reactive oxygen species (ROS). Excessive production of reactive oxygen species (ROS) can override antioxidant defenses, produce oxidative stress and DNA damage that triggers apoptosis and necrosis in organisms. However little is known about the harm of ROS induced by TMX during oocytes maturation. Here, bovine germ-vesicle (GV) oocytes were cultured to metaphase of the second meiosis (MII) stage in vitro with or without TMX. During this process, oocytes were evaluated by various methods. Microscopic examination showed that 1.6 mM TMX significantly inhibited the maturation process in which oocytes were arrested before MI stage or between MI and MII stage. Correspond to this two periods, immunofluorescence staining and enzyme activity analysis showed that active CDC25 and CDC2 reduced in TMX group compared to control; time lapse and immunofluorescence staining gave results that Cyclin B could not be degraded, actin cap could not form, and Bub3 could not be removed from kinetochores. In addition, MII oocytes exposed to TMX showed disordered chromosomes and spindle. To study further, oocytes cultured for 24 h were analyzed. On the one hand, these oocytes in TMX group accumulated more ROS and produced significantly decreased mitochondrial membrane potential and increased apoptotic signal compared to control by methods of quantities for dichlorodihydrofluorescein diacetate (DCHFDA), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide and Annexin-V, but the level of γH2AX protein in TMX group did not decline significantly compared with control. On the another hand, these oocytes were activated to be parthenogenetic embryos and cultured. Assessment for embryo development showed decreased rates of cleavage, morula and blastocyst in TMX group compared to control in vitro. In conclusion, these results suggest that ROS induced by TMX results in dysfunction of mitochondria and apoptosis, which block bovine oocytes to MI stage, trap them at AI/TI stage and trigger disordered chromosomes and spindle at MII stage. Additionally, MII oocytes with poor qualities result from TMX lose abilities to cleavage and develop to be morulae and blastocysts.

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58 ± 8.31 and 13.25 ± 7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75 ± 1.98 vs. 8.23 ± 6.07, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

One of the major hallmarks of uterine diseases is disruption of ovarian steroid hormone control of uterine cell proliferation and differentiation. Estrogen (E2) stimulates proliferation of uterine epithelial cells while progesterone (P4) is inhibitory to E2-mediated proliferation of the epithelium. Mitogen inducible gene 6 (Mig-6) is an important mediator of P4 signaling to inhibit E2 signaling in the uterus. Uterine-specific knockout of Mig-6 caused endometrial P4 resistance and infertility. Levels of ErbB2 (also known as HER2) and phospho-ERK1/2 are significantly higher in Mig-6 knockout mice as well as infertile women with endometriosis. To determine the interplay between Mig-6 and the Erbb2 signaling pathway in the uterus, we generated mice with Mig-6 and Erbb2 conditionally ablated in progesterone receptor-positive cells (Pgrcre/+ Mig-6f/f Erbb2f/f; Mig-6d/d Erbb2d/d). Mig-6d/d mice were infertile whereas control and Mig-6d/d Erbb2d/d mice exhibited normal fecundity. The uterine horns of Mig-6d/d mice had no implantation sites, whereas control and Mig-6d/d Erbb2d/d mice had averaged implantation sites. Additionally, aberrant increment of epithelial proliferation in uterus of Mig-6d/d mice did not show in Mig-6d/d Erbb2d/d mice uterus at pre-implantation stage. Microarray analysis revealed that almost altered genes in Mig-6d/d mice were recovered their expression levels in Mig-6d/d Erbb2d/d mice. The altered pathways such as cell-cycle control, DNA replication, and modification processes by Mig-6 ablation were rescued in Mig-6d/d Erbb2d/d mice. The infertility seen in Mig-6d/d mice is recovered in Mig-6d/d Erbb2d/d mice. These results suggest that Mig-6 mediates a critical P4 function to inhibit E2 signaling by inhibiting ErbB2 signaling. As MIG-6 is a mediator of P4 signaling, the activity of which can suppress unopposed-E2 signaling, our studies provide a potential new drug target for the intervention of female infertility.

Lately, CRISPR-Cas9 has become one of the most essential tools to understand gene’s function. In the honeybee, however, the application of CRISPR technology has been hindered by various factors leading to very few reports of success in genome editing. Among these, collection of honeybee embryos for microinjection has been a time-consuming procedure, mainly limiting the applicability of the genome editing technique to honeybees. To improve the drawbacks of the conventional plastic plug-based system, we have developed a film-assisted honeybee embryo collection system (FECS) using transparent film as a detachable bottom layer. In this new system, eggs are laid on the detachable film surface and collected in a batch, and thus no additional alignment is required for microinjectoin. As the film can be easily replaced with in a few seconds, embryo collection can be repeated continuously after a single caging of a queen. Also, unlike conventional plug-based systems, the new system utilizes 100% of the eggs laid by the queen, thereby increases the yield three times in theory. The main unit of the system can be printed with ordinary SLA/DLP type 3D printer and the stl file for 3D printing will be distributed online.

This study is to compare the effect of estrus synchronization and embryo transfer between Korean and Mongolian cattle. Embryos were collected from 9 donors housed in Asan city in South Chungcheong Province, South Korea. Embryos were collected 9 donors from Khushaat sum, Selenge province and Bayanchandmani sum, Tov province in Mongolia. Follicle Stimulating Hormone (FSH), Controlled Internal Drug Release (CIDR) and Prostaglandin (PG) were used for superovulation. Subsequently, Artificial Insemination (AI) was done for donor cow and embryo was collected after 7 and 8 days. Collected embryos were compared between Mongolian and Korean cattle. Finally, good quality and fresh embryos were transferred to 50 and 22 recipients of cows in Korea and Mongolia respectively. The findings show that Korean native cattle each donor cow produced on an average 16.9 embryos and, 10.9 embryos were found transferable. But in case of Mongolia the average production of embryos per donor cow was 8.6 embryos and, 6.2 embryos were found transferable. Embryo collection after 7 and 8 days was not difference in embryo production in Korea. But, in Mongolia embryo production after 8 days was found more efficient than the 7 days. Korean native recipient's cows (74.6%) and Mongolian recipient's cows (71.0%) respectively were found transferable ovarian stage. The result suggested that efficiency of embryo production from the superovulation method treated of Korean cow were higher than the Mongolian cow. The pregnancy rate of Korea native cattle was 72%, which was about 10% higher than that of Mongolia cattle.

An investigation of the effects of Pb for domestic anuran embryos were evaluated with the Frog Embryo Teratogenesis Assay; Xenopus (FETAX). Depending on the species, the difference between the embryo size and the embryonic development time was determined. As a result, mortality and malformation rates were increased, malformation patterns were changed and larval body length were decreased in a dose dependent manner of the Pb. The half maximal lethal concentration (LC50) of the Bufo gargarizans, Hyla japonica, Rana nigromaculata and Bombina orientalis were 0.58, 0.49, 0.52, 0.54 mg L-1, respectively. The half maximal effective concentration (EC50) of the Bufo gargarizans, Hyla japonica, Rana nigromaculata and Bombina orientalis were 0.35, 0.74, 0.30, 0.29 mg L-1, respectively. The teratogenic index (TI) were 1.66 in the Bufo gargarizans, 1.81 in the Hyla japonica, 1.73 in the Rana nigromaculata and 1.86 in the Bombina orientalis, respectively. Therefore, the Pb seems likely to have a teratogenic effect in all four species’ embryonic development. The Bombina orientalis was the most sensitive to the Pb. This means that the difference between the different species, even if they have all been exposed to the same concentration of pollutants depending on the species. The result above show that the Pb acts as a teratogenic agent in the development of the four domestic frog species.

Commercial applications of OPU/IVP were to produce embryos and calves from high genetic cows.The aim of this present study was to compare the number of recovered oocytes and cultured in vitro produced embryos from Ovum Pick-up (OPU). OPU derived embryo production was carried out of oocytes by ultrasonographic guided follicular aspiration and then produced in vitro produced blastocysts by IVP culture system. In result, the rate of recovered oocytes was obtained 612 (57.2%) and 451(73.7) G1+G2 grade oocytes. No difference of recovered rate (51.1~62.1%) was seen in six donor. The rate of cleavage and blastocyst development were obtained 320 (70.9%) and 78 (24.4%) that was 3.3±0.4 cleaved embryo and 0.9±0.2 blastocysts per session. Cleavage rate of OPU oocytes in No. 6 donor was 90.6%, significantly (P<0.05) higher than that in the other donors, However, blastocysts was similar (25.8~30.0%). In conclusion, limited numbers of OPU oocytes had competent development when cultured in SOF culture medium

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in Vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in Vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in Vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in Vitro maturation of pig oocytes.

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-17ß (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 μm, 814 μm and 1,200 μm. The serum P4 and E2 concentrations were 7.91±0.37 ng/μL and 45.45±12.65 ng/μL in the donor mare, and 16.06±3.27 ng/μL and 49.13±10.09 ng/μL in the recipient mare.

Embryo development is very important in reproductive physiology of domestic animal experiments. Therefore, in the above experiment, we want to provide a lot of important information with regard to fertilization breeding by looking at the expression of transcription factor by early embryo development. It is known that mice affect early embryonic development of many transcription factors, many experiments are underway. Different types of mammals showed different expression patterns, thus, we used pigs, which are known to be the most similar to humans, to observe the expression of transcription factors in early embryonic development. Transcription factors were observed using CDX2, OCT4 and E-CADHERIN. CDX2 was expressed in 2 cells, OCT4 and E-CADHERIN were expressed in blastocyst. OCT4 was expressed specifically in ICM (inner cell mass) in blastocyst, and E-CADHERIN was expressed in cell wall and junction of blastocyst. These results show that CDX2, OCT4 and E-CADHERIN play an important role in early embryonic development in pigs.

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

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.

The OPU technology has been largely used in order to enhance genetic improvement in domestic animals. This study demonstrated that OPU in Hanwoo can be used for the effective technique to improve the reproductive efficiency. This experiment showed the longest times of OPU ever carried out in Hanwoo. In this study four donors were selected from Hanwoo by using DNA extraction and SNP maker. Individual donors have genes which are CAPN1, CAST and FASN and that contain dominant position. CAPN1, CAST genes are often thought to be related with tenderness and FASN gene is thought to be associated with oleic acid which is identified a monounsaturated fatty acid found naturally in many plant and animal products. In experiment 1, OPU technique was used to evaluate the influence of the number of oocytes recovery rate per session. Totally 50 times OPU sessions were performed and oocytes recovery at every 10 times session was evaluated. In case of H4, the OPU session could be done around 30 times after her calving. Compared to the average number of oocytes recovery, H1 was more efficient than H3. Considering this results, the current study showed that animals have considerable individual variation in numbers of oocytes. In this study, the average recovery rate in Hanwoo is similar to the recovery rate of the Bos Taurus. Individual donors have no significant difference among group for recovery rate during 30 sessions. However, Thoese showed significant decrease in the number of oocytes recovery rate after 40 session of OPU treatment. Therefore we conclude that the Hanwoo donor is considered to be suitable for 30 times of OPU treatment. In experiment 2, OPU technique was used to evaluate the influence of the number of recovery rate monthly. The average number of oocytes recovery rate showed no significantly difference for five months. However, the number of oocytes recovery rate decreased significantly during three months after first five months experiments. In experiment 3, whether donors are parity or non-parity, the average number of oocytes recovery rate was checked. However, we could not find any significant result from this experiment. In experiment 4, the developmental rate of in vitro produced embryos from OPU was compared with that from slaughterhouse. From this, cleavage rate of oocytes from OPU is significantly less than that from slaughterhouse. In conclusion, this study included a thorough analysis of oocytes and embryo production by OPU from Hanwoo. OPU can be successfully performed under a continuous regime for 8 month in Hanwoo. The current study shows the clear proof that OPU could be adopted to produce oocytes and embryos of better quality as an advanced technique replacing the usage of MOET in Korea. Finally, elucidation the basis for numerous oocytes obtained from Hanwoo may contribute to a better understanding of reproductive physiology in cattle.