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Genetic resources of buffalo, cattle, goat, sheep, deer, rabbit, pig, chicken, duck, goose, turkey and swan in Taiwan are conserved in living form or frozen genetic materials. Preserving farm animals in living form must conserve a considerable number of mature individuals with reproductive potential, and continued funding and breeding space are necessary. Based on the afore‐mentioned restraints and consideration of risk diversification, living animals are preserved in Taiwan Livestock Research Institute (TLRI) branches and breeding stock reproduction grounds throughout Taiwan. Genetic materials preserved in frozen form include germ cell (sperm, egg and embryo), tissue, somatic cell, cell line, DNA and gene pool. Those preserved for long term more than 10 years are conserved in liquid nitrogen storage tank at ‒185 to ‒196℃. Animal industry on utilization of farm animal genetics emphasizes the importance of in situ conservation and considers ex situ conservation as an essential complementary activity to in situ. In situ and ex situ utilization are complementary, not mutually exclusive. The exact strategy of germplasm cryobanking will clearly depend on the conservation objectives of TLRI. In situ and ex situ strategies differ in their capacity to achieve the different bio‐utilization objectives. Based on the objectives for bio‐utilization and conservation, the existing national technical capacity and infrastructure for cryoconservation, and amount of capital to invest in developing and maintaining a gene bank for food animal genetic resources (FAnGR), each country should determine for which FAnGR, if any, national cryobanking program should be undertaken. The health and sanitary issues of animals that must be considered when establishing and operating gene banks for animal genetic resources to help prevent the conservation of potentially dangerous pathogens along with the valuable genetic material in the ultra‐low temperature cell repository. Bio‐utilization of material stored in the bio‐bank must eventually be thawed and used to create new animals and therefore, national organization and annotation of the stored material is critical to ensure its proper utilization. Stock animals are the source of semen, embryo and oocytes are usually privately owned. This ownership may or may not change during the gene banking process, but the terms of agreements between bio‐banks and donors must be explicitly defined. Taiwan Animal Germplasm Center now conserves genetic resources of 77 livestock breeds and strains, including 19 native and 38 foreign species as well as 20 new breeds produced by inbreeding in poultry or breed‐crossing with the artificial insemination in livestock. Cryobanking of semen of native animals is for genetic diversity but also for risk management to long‐term global food security. Ex situ conservation program involving in vitro storage of germplasm cryobanking can contribute to ensure that it will be available to allow livestock keepers and animal breeders to confront future changes in animal production and economic environments.

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).

Embryo transfer has been used in Japan for several years to produce bulls and cows of high genetic value, to produce beef calves from dairy cows. The average size of Japanese cattle farming is not very large. An efficient embryo transfer program is important to facilitate adoption of these technologies in the field. The fixed‐time embryo transfer programs allow for systematic embryo transfer under field conditions. The objective of this paper was to evaluate the practical utility of fixed‐time embryo transfer programs in cattle under field conditions. Two fixed‐time embryo transfer programs were used for dairy or beef cattle: 1) the ovysync program and the 2) progesterone and estradiol program. 1) Ovysync Program Dairy cattle (cows, n = 146; heifers, n = 107) were randomly allocated to a natural estrus control group (cows, n = 63; heifers, n = 47) or an ovulation synchronization (ovysync) group (cows, n = 83; heifers, n = 60), which was treated with an intramuscular (IM) injection of 100 μg GnRH at a random stage of the estrus cycle. Seven days later, the cattle received PGF2α (Cows; 25 30 mg) or PGF2α analog (Heifers; 0.5 mg) to regress the corpora lutea (CL). Forty‐eight hours later, the cows and heifers received a second injection of 100 μg GnRH. Embryo transfer was carried out 6 or 7 days after the second GnRH injection. There were no differences in the proportion of acceptable embryo transfers in the control (cows, 81.0%; heifers, 91.4%) and ovysync groups (cows, 83.1%; heifers, 91.7%). Pregnancy rates did not differ between groups. 2) Progesterone and Estradiol Program All beef heifers and beef or dairy cows received CIDR and estradiol benzoate (EB, beef heifers and cows, 1 mg; dairy cows, 2 mg) IM on Day 0, PGF2α at the time of CIDR removal (beef heifers and cows, Day 7; dairy cows, Day 8), 1 mg EB IM on Day 8 (beef heifers and cows) or 9 (dairy cows). Embryo transfer was carried out on Day 16 (beef heifers and cows) or Day 17 (dairy cows). The pregnancy rates were 80.0% (12/15) for beef heifers, 46.7% (7/15) for beef cows and 68.4% (13/19) for dairy cows. These results suggest that both fixed‐time embryo transfer programs can be effectively applied to cattle programs under field conditions.

<Objective> Injection of a linear transgene into male pronucleus has been widely used to produce Transgenic (Tg) mice. This approach however is inefficient and results in concatemerised transgene insertion and associated reduced protein expression from such insertions. The objective of this study is to develop active transgenesis method by using a piggyBac transposase plasmid DNA, and generate double transgene harboring transgenic mice. <Method> We examined the piggyBac transposase plasmid (pm- GENIE‐3) on its ability to produce transgenic animals with NaOH, HCl and FuGENE6 treated sperm followed by ICSI‐Tr, for its effectiveness in creating EGFP Tg mice, as judged by offspring epifluorescence. After these steps, we explored if the embryo development affects ICSI‐Tr efficiency by using substrate‐free media or aphidicolin. Moreover, we tested to determine if transgenesis is possible by directly injecting the DNA into the cytoplasm or into pronuclei. Finally, we attempted the introduction of two transgenes, such as EGFP and dsRED simultaneously in one transposon and the ability to generate double Tg mice by using NaOH treated sperm during ICSI‐Tr. <Results> The best results were obtained when sperm were treated with NaOH and co‐incubated with circular plasmid DNA of pmGENIE‐3. This resulted in Tg pups that could successfully express EGFP, with efficiencies of 37.9% of born animals being transgenic. Furthermore, the effectiveness of this method was proved by the production of Tg offspring from inbred strains of mice, such as C57BL/6, Balb/c and CD‐1 nude. While injection of DNA into the pronucleus or cytoplasm of one cell embryos, and delayed embryo development‐method were not as effective as ICSI‐Tr in producing Tg mice, they nevertheless proved successful. Finally, NaOH‐ICSI‐Tr successfully obtained Tg mice expressing both the EGFP and dsRED transgene. In conclusion, the current study developed an active form of NaOH‐ICSI‐Tr mediated transgenesis utilizing the piggyBac transposition machinery, and was successful in obtaining Tg mice which expressed simultaneously not only EGFP but also the dsRED transgene stably inserted in these animals.

The circling (cir/cir ) mouse is a murine model for human non‐syndromic deafness DFNB6. The causative gene is transmembrane inner ear (tmie), in which the mutation is a 40‐kilobase genomic deletion including tmie. The function of Tmie is unknown. To better understand the function of Tmie, we observed the spatiotemporal expression of tmie in the mouse cochlea using a Tmie‐specific antibody during postnatal inner ear development. Tmie was expressed in the cochlear hair cells of the mouse inner ear from embryonic days to adult. It is postulated that Tmie protein is involved in the hair cell structural formation and maturation before hearing onset (around P14), and maintenance of organ of Corti tissues after that. The cochlear hair cells of the circling mouse showed a basal‐to‐apical gradient of outer hair cell degeneration. The hair cell stereocilia bundles revealed the abnormal structure and it expanded to the apical region. In order to find the exact localization of Tmie protein inside the cell, we transfected the plasmids expressing GFP‐Tmie fusion protein into the HEI‐OC1 auditory hair cells. Tmie protein was colocalized with Calnexin (Canx), ER marker protein, but not with beta‐COP, Golgi marker protein. We next produced the Myo7a promoterdirected tmie expression transgenic mice to induce the phenotypic rescue of circling mice in a gene therapeutic way. Some circling mice with tmie transgene showed the normal behavior and hearing ability. These results indicate that tmie has a critical role in the inner ear development and hearing ability in the mice.

Active calcium transport is carried out by calcium channel proteins, cytosolic buffering or transfer proteins, and pump proteins. Several components of this transport system have recently been determined using gene knockout (KO) models. The calbindin‐ D9k/28k and calbindin‐D9k/TRPV6 double KO mice were generated and reported that induction of expression of some duodenal calcium transport proteins can compensate for the CaBP‐9k gene deficiency. In CaBP‐9k KO mice, the levels of these hormones differ between the KO and wild‐type (WT) mice. The induction of TRPV6 in the duodenum was observed in adult KO male mice but induction was not modified by physiologic doses of 1,25(OH)2D3 and compensatory gene induction was not affected by PTH. Duodenal TRPV6 transcription in WT and female KO mice were modulated by 1,25(OH)2D3 in a dose‐dependent manner. Under calcium‐deficient dietary conditions, in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of TRPV6 mRNA was significantly decreased in DKO mice fed a calcium‐deficient diet as compared to CaBP‐28k KO or WT mice, and DKO mice died after 4 weeks on a calcium‐deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25‐(OH) D3‐ eficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. Using microarray analysis, NCKX3 was identified as a gene that was differentially expressed in the kidneys of female and male mice. Although any hormones did not alter NCKX3 expression, however, aldosterone and hydrocortisone did down‐regulate renal NCKX3 expression in female mice. Taken together, these results indicate that deletions of CaBP‐9k and 28k has a significant effect on calcium processing under calcium‐deficient conditions, confirming the importance of dietary calcium and 1,25‐(OH)2D3 during growth and development

The composition of culture media is a key element in the process of in vitro embryo production. With the development of defined culture media, many components that are present in trace amounts in follicular fluid and serum have been excluded from the in vitro embryo production system. Among these are hormones, which have important regulatory roles in growth, metabolism and differentiation and are known to be present in follicular fluid, serum and the female reproductive tract. We have investigated the effects of supplementation of in vitro maturation and/or culture medium with testosterone (T), androstenedione (A4) and thyroid hormones (TH) on bovine in vitro embryo production and the mechanisms of action of TH in developing embryos. Our results show that testosterone, but not androstenedione or thyroid hormone supplementation increased cleavage rates. None of the treatments significantly altered the sex ratio. Addition of thyroid hormones, T3 and T4, to the in vitro culture media resulted in a significant increase in the rate of development to the blastocyst stage. In addition, blastocysts from the T3/T4 treated groups had higher cell number and lower rates of apoptosis. We have confirmed the expression of mRNAs for both Thyroid hormone receptor α and β (TR α and β) in cumulus‐oocyte‐complexes, oocytes, and in both treated and control blastocyst. Quantitatively, the expression of TR mRNA was higher in the treated embryos but the difference was not statistically significant. TR proteins were detectable in blastocysts of both groups with a difference in the distribution pattern. TH treated embryos had peri‐nuclear concentration of TR while in control embryos it was homogenously distributed in the cytoplasm. Preliminary studies of inhibition of TR α and β by siRNA knockdown by micro injection at the zygote stage show a drastic reduction in development suggesting that TH play an essential role in embryo development. They appear to mediate this effect by their receptors, TR α and β. Overall the results show that the presence of hormones in maturation and the culture medium can alter the outcome of in vitro embryo production and highlight the significance of biological components missing from in defined embryo culture media.

X‐box binding protein‐1 (XBP‐1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP‐1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP‐1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP‐1 was weak in mature oocytes and at the one‐cell, two‐cell, and eight‐cell stages of embryos, but abundant at the GV oocyte, four‐cell, morula, and blastocyst stages. In addition, RT‐PCR revealed that both spliced XBP‐1 (XBP‐1s ) and unspliced XBP‐1 (XBP‐1u) were expressed at the GV oocyte, four‐cell, morula, and blastocyst stages. Tunicamycin (TM), an ER stress inducer, blocked porcine embryonic development at the four‐cell stage, exhibiting the effect on embryonic genome activation. Next, porcine embryos cultured in the presence of tauroursodeoxycholate (TUDCA), an ER stress inhibitor, were studied. Total cell numbers and the extent of the ICM increased (p<0.05), whereas the rate of nuclear apoptosis decreased (p<0.05). Moreover, expression of the anti‐apoptotic gene Bcl‐2 increased whereas expression of the pro‐apoptotic genes Bcl‐xl and p53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress‐mediated apoptosis in vitro.

Successful pregnancy requires well-coordinated interactions between the maternal uterus and the developing embryo in pigs. In pigs, implantation begins around Day 12 of pregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes various factors such as estrogens, interleukin-1 beta (IL1B), and interferons. Estrogens produced by conceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogen action is explained by the endocrine and exocrine theory. The uterine endometrium becomes receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells and increasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone, progesterone, but the presence of conceptus in the uterus also induces changes of endometrial functions, including most importantly maternal recognition of pregnancy. Many factors, such as hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs. However, understanding of the cellular and molecular events occurring in the endometrium is not complete. In recent studies we made some progress on understanding of expression and function of genes involved in maternal-fetal interaction for the establishment and maintenance of pregnancy in the uterine endometrium in pigs. Firstly, we found that lysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of implantation and LPA receptor 3 was uniquely expressed in the endometrium during early pregnancy. Secondly, we observed that salivary lipocalin (SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time of embryo implantation, and its expression was regulated by IL1B. Furthermore, expression of IL1B receptors are regulated by estrogen and IL1B, and IL1B functions in expression of genes related to prostaglandin synthesis and transport. Thirdly, we found that calcium regulatory molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo implantation and the maintenance of pregnancy. Finally, we observed that an MHC class II molecule, SLA-DQ, is expressed in the uterine endometrium at the time of conceptus implantation and its expression is essential for successful pregnancy, indicating that appropriate maternal-fetal immune interaction is required for the maintenance of pregnancy. Further analysis of these molecules will provide insights into the cellular and molecular basis of maternal-and fetal interaction during pregnancy in pigs.

Pig‐to‐human transplantation (xenotransplantation) is currently the most advanced approach to solving the increasing demand for human organs and tissues. However, two critical requirements must be addressed before xenotransplantation can be considered for clinical application. First, the level of immunosuppression required to maintain xenografts must be equivalent to (or less than) that used in allotransplantation. It is now evident that multiple genetic modifications of the donor pig will be needed to achieve this goal (d’Apice et al. 2002 Transplant Proceedings. 33: 3053‐3054). These include gene knockouts (e.g. of the GalT gene, responsible for synthesis of the major porcine xenoantigen) and gene addition by transgenesis. Progress has been hindered by the current technology, which allows only a single cycle of genetic modification per generation and therefore necessitates large and complex breeding programs. Second, donor pigs should have defined, relatively homogeneous genotypes including the inability to produce endogenous retroviruses (PERV) that may infect human recipients. Inbred miniature swine are best suited in this regard but are difficult to genetically manipulate due to poor reproductive capacity. What is critically needed to advance xenotransplantation to the clinic is the ability to perform multiple cycles of genetic modifications per generation on the background of choice. We have recently made an important step towards this goal by developing a novel method for the isolation of porcine embryonic stem cells (ESC) (Vassiliev et al. 2010 Cellular Reprogramming 12: 223‐230). These cells can be stably grown for at least 150 population doublings, dramatically increasing the window for introducing multiple genetic modifications before the cells are used to clone pigs by somatic cell nuclear transfer (SCNT). Furthermore we have used this method to isolate ESCs from cloned embryos (Vassiliev et al 2011 Cellular Reprogramming 13: 205‐213) which allows us to isolate ESCs directly from breeds of pigs specifically bred for xenotransplantation. Together these advances will accelerate xenotransplantation research to the clinic.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

Spermatogenesis is a series of complex processes that produce spermatozoa in male testis and it occurs through consecutive cell divisions and differentiation of germ cells (Russell et al., 1990). This process is initiated by a small number of spermatogonial stem cells (SSCs) that are only two or three per 104 testis cells in mouse case, and finally gives rise to many functional spermatozoa. Similar to the characteristics found in other adult stem cells, SSCs have the capability of self‐renewal and differentiation (Meistrich and van Beek, 1993). SSCs that have these two capabilities are the source of maintaining male postnatal fertility for lifetime. SSCs that exist inside the male testis maintain the numerical equilibrium through self‐renew after birth and they are the only germ‐line stem cell that can transfer the genetic information to the next generation through spermatogenesis. Therefore, when genetic modification is performed at the SSC stage, it can produce transgenic offspring in the next generation as well as germ‐line modification so that it can deliver the transformed character stably to the descendants. Past studies regarding the SSC had been dependent on morphological observations due to the absence of a marker system that can distinguish the SSCs. Brinster et al. (1994) published a groundbreaking turning point in identifying characteristics of SSC by developing SSC transplantation technique (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). Utilizing the SSC transplantation technique, the self‐renew and production capability of differentiated tissue derived from transplanted SSC within the recipient’s seminiferous tubule can be directly analyzed. The biological activity of SSCs can also be investigated objectively by the SSC transplantation technique. Since the advent of the SSC transplantation technique, there have been a lot of progresses in the biological field of SSC. Recently, the enrichment technique of SSCs using FACS and specific surface marker, in vitro culture, and genetic modification techniques of SSCs have been developed in rodents. These techniques have potential to enhance the practical applications of SSCs. Characterization and development of useful technique for SSCs are now extending to livestock species.

Somatic cells achieve a pluripotent state by pluripotential reprogramming. During pluripotential reprogramming, somatic cells re-established various features of pluripotent cells, such as the expression of pluripotency markers, inactivation of tissue-specific gene expression, developmental potential to contribute to all three germ layers, and an undifferentiated epigenetic state. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. These iPS cells are potentially a valuable source of immune matched pluripotent stem cells that can be differentiated and used for tissue replacement therapies. Recent technical advance in direct reprogramming of somatic cells lead to a safe, viral- free iPS cell generation. Here we develop new techniques to generate iPS cells. Using titanium oxide (TiO2) nanotubes. we could successfully transfer reprogramming proteins (Oct4, Sox2, Klf4, Nanog and c-Myc) into somatic cells. After two weeks of treatment of protein conjugated nanotubes, somatic cells adopted an ES cell-like morphology and activated Oct4-GFP, which is pluripotency marker, indicating that nanotubes can be used for protein delivery carriers, which induce cellular reprogramming. Next, we induced differentiation of iPS cells into neural stem cells (NSCs) and compared with mouse embryonic stem (ES) cell-derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from NSCs from brain tissue and rapidly propagated in the presence EGF and bFGF, and stained positive for NSCs markers Nestin and Sox2. Moreover, these NSCs have capacity of differentiation into multiple cell lineages, such as neurons, astrocytes, and oligodendrocytes. Induction of pluripotency and directed differentiation of iPS cells into a specialized cell type hold considerable promise for regenerative medicine as well as basic research.

The vast majority of embryo generated by Assisted Reproductive Technologies (ART) do not result in a live offspring and a multiple birth is the single biggest health risk associated with human fertility treatment, and the used of frozen embryos increased for medical or personal reasons. However, practical and ethical reasons might hamper study of human embryos. Therefore, animal models are necessary to elucidate the molecular and morphological changes during development. In the serial experiments, we employed mouse embryos and a Cdx-inducible ES cell system that transdifferentiates into TS cells. We found aberrant gene expression profiles including apoptosis associated (Bcl2), lineage formation related genes (Cdx-2, Tcfap2c, Oct4, and Nanog), and/or mitochondrial DNA replication related genes (mt-cox-1, mt-cox-2, Polg, Polg2, Tfam) in mouse embryos that showed developmentally retardation between morula to blastocyst transition or post implantation development after embryo transfer to surrogate mothers, compared to control embryos. To determine direct interaction between knockdown genes via siRNA approach and putative down-stream genes involved in blastocyst formation and further development, we carried out qPCR and Chip assay in either mouse embryos or the ES cells. qPCR and Chip assay results showed target gene directly bound to promoter regions of down-regulated genes in TS cells. In conclusion, we suggested that an increased understanding of epigenetic regulation of early embryonic development through animal models may ultimately lead to better methodologies for selecting more competent embryos and and/or protocols for augmenting embryos viability.

Cloning or somatic cell nuclear transfer (SCNT) using adult somatic cell to derive cloned embryos is a promising new technology with potential applications in both agriculture and regenerative medicine. Mammalian embryos derived by nuclear transfer are capable of development to the blastocyst stage with a relatively high efficiency of 30～ 50%. However, in full-time development, usually only 2% of NT embryos can result in live births due to abnormalities in placenta formation. In SCNT embryos, the donor cell nucleus is epigenetically reprogrammed by oocyte cytoplasm during development. Incomplete reprogramming of the donor cell genome is considered a major reason for low cloning efficiency. Aberrant epigenetic modifications include DNA methylation, histone modification and X-chromosome-inactivation. Due to a lack of basic knowledge regarding the embryos following nuclear transfer, the success rate of cloning is low. Therefore, elucidation of the molecular mechanism of SCNT embryo development will be of great value for further research. MicroRNAs (microRNA) are single-strand RNA molecules of about 19 23 nucleotides in length, which regulate gene expression by imperfect base pairing with target mRNA, subsequently guiding mRNA cleavage or translational repression. Since the first discovery and functional annotation in 1993 of the small RNA, lin-4 and let-7, which are involved in developmental timing and gene regulation during C. elegans larval development, microRNAs have received scientific attention. Now hundreds of microRNAs have been identified in various multicellular organisms, and many microRNAs have been shown to be evolutionarily conserved. The roles proposed for this novel class of tiny RNA molecules are diverse. They are likely to be involved in developmental timing, differentiation, cell proliferation, signaling pathways, apoptosis, metabolism, heterochromatin formation, genome rearrangement, brain development and carcinogenesis. Currently (2006- present) we are working to determine the role of microRNAs on the epigenetic regulation of fertilized and cloned embryo development. The general hypothesis of our research is that genetic and epigenetic factors regulate the development of preimplantation mammalian embryos, and aberrant modulations in cloned embryos are causes of abnormal development and low success rate of cloned embryos.

In this study, we examined the effects of porcine granulocyte-macrophage colonystimulating factor (pGM-CSF) on in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) at first time. The objective of present study was to verify effects of pGM-CSF on SCNT-derived blastocyst formation and evaluate gene expressions and qualities of the blastocyst formed after pGM-CSF treatment. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. A total 522 cloned embryos in 6 replicates were treated with 10 ng/ml concentration of pGM-CSF during in vitro culture (IVC). It was demonstrated that treatment of 10 ng/ml pGM-CSF could increase blastocyst formation and total cell number in blastocyst significantly (p<0.05) compared to the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no any effect on cleavage rate. It was found that the number of cells in the inner cell mass (ICM) and trophectoderm (TE) were significantly increased compared to the control (4.4 and 31.9, respectively) when cloned embryos were cultured with 10 ng/ml pGM-CSF (6.0 and 43.0, respectively). It was also found that treatment of 10 ng/ml pGM-CSF significantly (p<0.05) increased POU5F1 and Cdx2 mRNA expressions in blastocysts. In addition, Bcl-2 mRNA expression was found to be significantly (p< 0.05) up-regulated in blastocysts in the pGM-CSF supplemented group compared to the control. In conclusion, these results suggest that pGM-CSF may improve the quality and developmental viability of porcine cloned embryos by enhancing nuclear reprogramming via regulating transcription factors expression.

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, gene expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). In the nuclear maturation after 44 h IVM, the groups of 0.1, 0.5, and 2.0 μM (83.0%, 84.1%, and 88.3%, respectively) had no significant difference compared to the control (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.

국내의 말 산업 확대와 승마인구 저변확대에 따른 승용말 생산의 필요성이 대두되고 있으나, 현재 대부분의 승용말은 경주 퇴역마 또는 고가의 수입말을 활용하고 있다. 따라 서 우수하고 경제적인 승용말의 국내 생산이 필요하며, 이에 따른 인공수정 및 수정란이 식 연구가 필요하다. 본 연구에서는 국내 환경에 적합한 말의 수정란 채란 및 이식 체계 를 확립하기 위하여 수행하였다. 공란말 및 수란말의 발정동기화는 초음파를 이용 난소검사 후 황체와 난포가 확인된 개체에 대하여 PGF2a를 근육주사 하였다. 발정이 확인된 개체는 난포크기를 측정하였고, 4 cm 이상에 도달하였을 때 배란유도 및 인공수정을 실시하였다. 수정란의 채란은 Equine Lavage Catheter (Bioniche, USA), 회수용 채란액은 ViGRO(Bioniche, USA)를 이용하 여 인공수정후 5, 6, 7 및 8일째에 실시하였다. 채란된 수정란은 5% FBS가 첨가된 TCM- 199에서 38.5℃, 5%CO2 하에서 배양하면서 크기를 조사하였다. 공란말 8두에 발정동기화를 유도하여 6(87.5%)가 발정이 유도 되었고, 6두가 배란유도 및 인공수정에 성공하였다. 인공수정 공란말 중 5두가 채란에 이용되어 3(60%)두가 채란 에 성공하였다. 채란된 수정란은 수정 후 배양 5일째 220 μm, 6일째 580 μm, 7일째 1.2 mm 및 8일째 2.2 mm로 발달한 후 부화하였다. 한편 채란된 7일째 수정란 2개를 수란말 2두에 이식하여 1두가 임신에 성공하였다. 한편 공란말의 혈중 프로제스테론 및 에스트 로젠 농도는 각각 평균 7.91 ng/ul 및 24.45 pg/ul이었고, 수란말의 혈중 프로제스테론 및 에스트로젠 농도는 각각 평균 16.06 ng/ul 및 49.13 ng/ul였다. 본 연구를 통하여 국내에서도 말의 수정란 채란 및 이식에 성공하여 번식기술을 활용 한 말의 증식 및 개량이 가능할 것으로 생각된다.

Ovulation resembles a tissue remodeling process such as a blood coagulation. The present study was aimed to examine the involvement of tissue factor, a primary factor for extrinsic coagulation pathway, in the ovulation. Northern blot analysis revealed that mRNA levels of tissue factor and tissue factor pathway inhibitor 2 (TFPI-2) in the ovary were stimulated by human chorionic gonadotropin (hCG) treatment in surperovulation model, of immature rats. Real-time PCR analysis demonstrated that the expression of tissue factor and TFPI-2 was stimulated in granulosa and theca cells of preovulatory follicles, respectively. The induction of tissue factor mRNA was blocked by the progesterone receptor antagonist RU486. Tissue factor protein was not detected in the ovary by Western blot and immunohistochemical analysis due to the lack of a specific antibody. Interestingly, the levels of tissue factor and TFPI-2 mRNA were increased in the ovarian cells of rats induced ovarian hyperstimulation syndrome (OHSS) and in granulosa cells of OHSS patients undergiong in vitro fertilization. The present findings indicate the stimulation of tissue factor system during ovulation, and in OHSS patients, implicating the possible involvement of tissue factor system in OHSS.

국외에서 승용말 번식에서 인공수정 및 수정란이식의 활용도가 높지만, 국내에서 사육 되는 말은 대부분이 경주마로 자연종부로만 번식을 하고 있다. 인공수정 등 번식기술의 국산화를 위해서는 발정주기의 난포 변화와 난포 변화에 따른 호르몬 변화 등의 연구가 필요하다. 본 연구에서는 말 발정기의 난포크기에 따른 프로제스테론과 에스트로젠의 변 화 및 난포 크기에 따른 배란유도와 인공수정 결과를 조사하였다. 말의 난포 크기가 발정증상 개시부터 2 cm, 3 cm 및 4 cm 이상에서 혈액내 프로제스 테론과 에스트로젠을 분석하였다. 발정개시 후 난포의 크기에 따라 hCG를 이용 배란을 유도하였고, 배란 유도 후 24 및 40시간에 동결정액으로 2회 인공수정 하였다. 발정기의 말에서 혈액내 프로제스테론 농도는 난포의 크기가 각각 2 cm, 3 cm 및 4 cm에서 평균 9.09 ng/ml, 13.04 ng.ml 및 1.40 ng/ml로서 4 cm에서 급격하게 감소하였 다. 한편 에스트로젠농도는 각각 평균 59.20 pg/ml, 48.5 pg/ml, 및 41.07 pg/ml로서 차이 가 없었다. 배란유도 시점의 난포 크기가 임신율에 미치는 영향을 조사한 결과 난포 크 기가 4 cm 미만에서는 임신율이 0%였으나, 4 cm 이상에서는 60%였다. 배란유도 후 인 공수정 시점의 난포 크기가 임신율에 미치는 효과를 조사한 결과 난포 크기가 5 cm 미 만은 임신에 실패하였으나, 5 cm 이상에서 60%의 임신율을 나타냈다. 본 연구를 통하여 발정기 말의 배란유도 및 인공수정 시점에 주기적인 초음파 검사를 통한 난포 크기의 확인이 인공수정의 임신율에 영향을 미치는 것으로 판단된다.

한우 인공수정용 액체질소 공급이 월1회로 정액과 같이 공급되고 있어 특히 고온 계절 에는 질소 탱크에 액체질소가 항상 일정하게 유지되고 있지 않은 실정이며 유통과정 중 에 외기에 노출되는 기회가 많이 있음으로써 동결정액의 품질저하가 쉽게 발생된다. 생산 후 유통과정에 발생되는 동결정액의 품질 평가 시기존방법의 단점인 스트로 폐기와 장비 구입비 및 검사 소요시간을 보완하고 동결정액의 품질 변화가 일어나는 ‒160℃ ‒35℃ 사이의 품질을 조사할 수 있게 하여 유통과정상의 소동결정액의 품질저하를 방지 하고 품질 평가를 실시하기위하여 제작하여 조사 하였다. 가축 인공 수정소에서 소동결 정액이 ‒100℃에 노출되는 기회가 56.5%, ‒35℃에 노출은 8.7%가 발생되었으며, 이러한 결과는 동결정액의 생존성을 ‒100℃에서 노출 될 때는 26.9%, ‒35℃에서 노출될 때는 ‒54.4%나 저하됨으로써 농가 인공수정 수태율 저하를 일으키는 주요한 원인이 되고 있 는 것으로 조사되었다.

본 연구는 도축된 한우의 황체난소, 낭종난소, 정상난소를 이용하여 체외 수정란 생산 시, 각각의 난소별 수정란 생산율을 분석하기 위하여 각 난소별 미성숙 난자의 회수율을 조사하였다. 15 18℃로 운반된 도축 유래 난소로부터 18G 주사침이 장착된 10 ml 주사 기를 이용하여 적색체 또는 황체가 형성된 난소, 직경 15 mm 이상의 낭종성 난소 및 4 10 mm의 정상난소로부터 미성숙 난자를 회수하여, 체외수정을 통한 분할율을 확인하 였다. 총 회수된 미성숙 난자와 이용 가능한 미성숙 난자의 수를 조사한 결과, 황체난소, 낭종난소 및 정상난소에서 총 회수된 미성숙 난자는 18.94±1.11, 18.57±1.42 및 20.24± 2.28개로 관찰되었으며, 그 중 성숙 배양에 이용된 미성숙 난자는 7.92±0.77, 8.22±1.31 및 10.70±1.02개로 확인되었고, 모두 유의적인 차이는 관찰되지 않았다(p<0.05). 또한, 난 소에서 총 회수된 미성숙 난자에 대한 성숙 배양으로의 효율은 황체난소, 낭종난소 및 정상난소에서 각 42.39±5.01, 43.60±5.15 및 53.55±3.19%로 조사되었으며 유의적인 차 이는 나타나지 않았다(p<0.05). 한편, 황체난소, 낭종난소 및 정상난소로부터 회수된 미성 숙 난자의 분할율은 77.46±1.60, 74.91±2.03 및 77.39±2.39%로 관찰되었고 유의적인 차 이는 나타나지 않았다(p<0.05). 본 연구의 실험 결과 황체 및 낭종난소와 정상난소에서 회수된 미성숙 난자의 개수 및 분할율에는 차이가 없다는 것을 알 수 있으며 이와 같은 결과를 통해 황체 및 낭종난소에서도 정상적인 난소에서와 마찬가지로 미성숙 난자가 채 란되며 이용가능하다는 것을 알 수 있었다. 하지만 보다 정확한 결과를 얻기 위해서는 추가적으로 성숙 및 발달율에 대한 조사가 이루어져 할 것이라고 생각된다.

The study investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and gene expression. Three experiments were conducted. Firstly the trans-ε-viniferin was purified from the leaves and stems of the Vitis amurensis , a common wild grape found in Korea, Japan, and China. In the first experiment, a total of 594 cumulus oocytes complexes (COCs) were used for the evaluation of the nuclear maturation. COCs were matured with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM). After IVM 42 44 h, the nuclear maturation was evaluated. In the second experiment, a total of 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular GSH and ROS levels. In the third experiment, the gene expression of oocytes matured with trans-ε-viniferin (0.5 μM) and the untreated group were evaluated after IVM. As results, we observed that trans-ε- viniferin treatment during IVM did not improve the nuclear maturation. But significantly increased (p<0.05) intracellular GSH levels in 0.5 μM group (0 μM vs. 0.5 μM; 14.6 vs. 16.8 pmol/oocyte) and reduced ROS levels (0 μM vs. 0.5 μM and 50 μM; 174.6 vs. 25.7 and 23.8 pixel/oocyte). The trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (p<0.05) expression of Dnmt1 mRNA in 0.5 μM treatment group than in the control group. But, the other gene expressions (PCNA, OCT4, caspase3, BAK, BAX and sit1) did not significantly differ from the control. In conclusion, these results indicated that the trans-ε-viniferin treatment during porcine IVM increased the cytoplasmic maturation through increasing the intracellular GSH synthesis, reducing ROS levels and increasing the Dnmt1 gene expression.

Artificial insemination and embryo transfer is one of the most important factors affecting to the production of fawn from deer nuclear transfer in the field of deer farms. This study* was conducted to establish the production technology of nuclear transfered embryo in deer. For estrus synchronization or superovulation tretments in flower deer and elk, each 10 does were inserted into the vagina for 14 days with CIDR (Pfizer New Zealand Ltd., NZ) for elk and Ring-CIDR (Bioculture Co., Ltd., Korea) for flower deer, and then those inserted devices were removed. The estrus synchronization of each 6 does were induced by the intramuscular injection of PGF2α (25 mg/head) and PG600 (hCG 200IU + PMSG 400IU, Intevet, Holland). Then, the superovulation of each 4 does of flower deer and elk was induced by additional injection of FSH (200 mg/ head) twice with an interval of 24 hours , respectively. Follicular oocytes were collected from each 2 does superovulated after 48 hours since the injection of PG600 and FSH. In the meantime, the ovarian response and the number of the collected ovarian follicles were investigated with the surgical operations. As a result, the average number of the collected ovarian follicles were 8.5 and 9.0 in flower deer and elk, respectively. The ovarian follicles collected from each two does were cultured in vitro for 48 hours with m-DMEM medium, and then the cell fusion was carried out after the nuclear transfer by the antler cell. As a result, 5 out of 18 ovarian follicles collected from 2 elk does were reached on the MII stage, but there was no generation resulting from the nuclear transferred embryos by the antler cell after enucleation. In 2 flower does, 7 out of 17 ovarian follicles were reached to the MII stage, but one of them was developed to parthenogenetic embryo as well despite a case of fusion from the nuclear transferred embryo. Embryos were collected in a surgical way on the 7th day after artificial insemination, numbers of average embryos collected were 2.5 and 3.0 in each 2 flower deer and elk does superovulated, respectively. The collected two embryos were transplanted to each 2 does synchronized. As a result, a head of fawn was produced from only one elk doe, where as a head of fawn were delivered from one out of 4 elk does artificial inseminated. Given these findings, we consider that more or less of problems might have occurred in vitro culture system of ovarian follicles in the production of nuclear transfered deer embryos. In addition, the greatest reason why both the aetificial insemination and embryo transfer failed was considered attributable to stress due to anesthesia and catching.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

최근 국내 한우의 사육은 전업화 및 대규모화에 따른 전반적인 우군 번식관리의 부실 에 기인한 수정회수의 증가로 번식률 저하가 문제가 되고 있는 실정이다. 따라서 한우 암소의 산차가 수정회수에 미치는 영향을 구명하고 저, 충남 아산시 소재 대규모 한우 농장에서 사육되고 있는 연 2,193두를 대상으로 2008년부터 2010년까지 3 년간의 번식기록을 분석하였다. 연 2,193두에서 모든 산차(0 9산)에서 1회 수정두수가 1,525두, 2회 505두 순으로 나 타났으며, 3회 이상 수정한 저수태우는 163두로 7.4%를 차지하였다. 1회수정은 미경산우가 304두로 가장 높았으며 4산차 248두, 3산차가 228두 순으로 나 타났으며, 2회 수정은 미경산우가 107두로 가장 높았으며, 4산차 83두, 3산차가 79두순 으로 나타났다. 3회 수정은 미경산우가 44두로 가장 높았으며, 3산차가 21두, 2산차가 18두 순으로 나 타났고, 4회 수정은 미경산우가 13두로 가장 높았으며 3산차가 2두였고, 5회 수정은 미 경산우가 2두로 각각 나타났다. 이상과 같은 결과로 미루어 모든 산차에서 1회 수정이 1,525두로 69.5%였으며, 산차가 높을수록 수정회수가 감소하는 경향을 나타났으며, 3산차부터 5산차까지 2회 이상 수정 두수가 229두로 45.3%로 많은 비율을 차지한 것은 암소 번식우에 대한 철저한 발정관찰 과 번식관리가 필요할 것으로 사료된다.

기후변화와 동반하여 국내 한우 사육에 있어 생리적, 환경적 변화로 무발정과 미약발정 현상이 잦아지고 있으며, 번식효율은 매년 저하되고 있는 실정이다(Roche 등, 2000; Lucy, 2001). 또한, 발정발현이 줄어들고 있으며, 배란지연 수태율 저하 등 번식에 있어 서 여러 가지 문제점들이 대두되고 있다(Pankowski 등, 1995; Austin 등, 1999). 본 연구 는 한우 공란우의 이식가능한 수정란 수와 소의 영양 상태에 영향을 미치는 혈장내 요소 태질소(BUN), Glucose 및 Cholesterol 수준과의 관계에 대해서 조사하였다. 한우 55두에 대하여 발정주기에 관계없이 CIDR를 질내에 삽입 후 4일째부터 성선자극호르몬(안토린) 28AU를 4일간 12시간 간격으로 근육주사 하였다. 투여 3일째 CIDR를 제거하였으며, 동 시에 PGF2α 25 mg을 근육 주사하여 과배란을 유기하였다. 공란우의 인공수정은 PGF2α 투여 후 발정을 확인하고 12시간 간격으로 2회 인공수정 하였으며, 1회 인공수정 전 GnRH 100 μg을 근육 주사하였다. 수정란 회수는 1차 인공수정 후 7 8일째에 비외과적 방법으로 채란하였다. 한우 공란우 55두 중 황체수가 10 이하와 10 이상인 공란우의 과 배란 처리하여 수정란을 회수한 결과, 10 이하와 10 이상인 공란우의 총 회수 된 난자수 는 각각 8.9개와 14.3개 였으며, 이식가능 수정란수는 황체수가 10 이하인 경우 4.8개, 10 이상인 경우 5.6개로 나타났다. Glucose 수준이 60 mg/dl 이하일 때 총 회수된 난자 수는 7.3개로써 60 70 mg/dl과 70 mg/dl 이상일 때보다 비해 적게 나타났다. 이식가능 수정란 수에서도 60 70 mg/dl과 70 mg/dl 이상일 때에 비해 60 mg/dl 이하일 때 4.0± 1.3개로 5.9±1.9, 5.6±2.0개에 비해 낮게 나타났지만 유의적인 차이는 보이지 않았다. Cholesterol 수준에 따라 이식가능 수정란 수에서는 유의적인 차이를 보이지 않았다. Cholesterol 수준이 150 mg/dl 이하, 150 200 mg/dl 및 200 mg/dl 이상일 때 총 회수 난자 수는 11.5개, 12.2개 및 10.9개를 회수하였다. 또한, 이식가능 수정란 수는 각각 5.1±1.7 개, 5.4±1.3개 및 5.2±1.9개를 나타냈다. BUN 수준이 ＜10, 11 18 및 ≥19 mg/dl일 때 이식가능 수정란 수는 각각 4.3±1.3, 5.8±1.8 및 4.7±2.1를 나타냈다. 공란우 55두 중 황 체수가 10 이하와 10 이상인 공란우의 과배란 처리하여 수정란을 회수한 결과, 10 이하 와 10 이상인 공란우의 총 회수 된 난자수는 각각 8.9개와 14.3개였으며, 이식가능 수정 란수는 황체수가 10 이하인 경우 4.8개, 10 이상인 경우 5.6개로 나타났다.

Freezing of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification of bovine expanded, hatched and SCNT embryos on the survival rate, apoptosis index and further development after thawing. Expanded, hatched and SCNT embryos were vitrified after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. Artificial shrinkage of the blastocyst was achieved after pushing a pulled pasteur pipet into the blastocoele cavity until it contracted. The shrunken and not shrunken embryos were exposed to cryoprotectant solution in 7.5% ethylene glycol-7.5% DMSOPBS with 20% FBS for 5 min. They were placed in a small volume of vitrification solution (15% ethylene glycol+15% DMSO+PBS+20% FBS+0.5 M sucrose) and plunged into liquid nitrogen on a cryotop. Then, after thawing, cryoprotectant was diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min. Under the optimal conditions, overall efficiency of the survival rate of bovine expanded, hatched, SCNT embryos in artificial shrinkage groups was higher compared with non-artificial shrinkage groups (p< 0.05). Especially, the numbers of TUNEL-positive nuclei in artificial shrinkage groups were significantly reduced than those of non-artificial shrinkage groups among frozen-thawed expanded, hatched, and SCNT blastocysts (p< 0.05). Our results showed that survival rates in cryopreserved expanded, hatched, SCNT embryos could be improved by reducing the fluid content. Therefore, we suggest that artificial shrinkage method is a effective pretreatment technique for the cryotop vitrification of expanded, hatched, SCNT bovine blastocysts.

한우 60두에 대하여 발정주기에 관계없이 CIDR를 질내에 삽입 후 4일째부터 성선자극 호르몬(안토린) 28AU를 4일간 12시간 간격으로 근육주사 하였다. 투여 3일째 CIDR를 제 거하였으며, 동시에 PGF2α 25 mg을 근육 주사하여 과배란을 유기하였다. 공란우의 인공 수정은 PGF2α 투여 후 발정을 확인하고 12시간 간격으로 2회 인공수정 하였으며, 1회 인공수정 전 GnRH 100 μg을 근육 주사하였다. 수정란 회수는 1차 인공수정 후 7 8일 째에 비외과적 방법으로 채란하였다. 총 60두를 공시하여 55두에서 발정이 유기되었으 며, 발정 및 승가는 유기된 55두 중 각각 2두, 34두 및 19두에서 발정 발현을 보였다. 발 정 유기율에서 BCS 2.25 2.75와 2.75 이상인 우군에서는 97.1% 및 95.0%인 반면 2.25 미만인 우군에서는 40.0%로 유의적인(p< 0.05) 차이를 보였으며, 승가율에서도 BCS 2.25 2.75인 우군에서 94.1%, 2.75 이상인 우군은 89.5%를 보인 반면 BCS 2.25 미만인 우 군에서는 50.00%로 BCS 2.25 이상인 우군에 비해 유의적(p< 0.05)으로 낮게 나타났다. 과배란 처리 우군의 발정 유기율, 승가율과 BUN 수준과의 관계에서는 10 mg/dl 이하 우 군, 11 18 mg/dl 사이 우군 및 19 mg/dl 이상인 우군에서 각각 77,8%, 95.1%, 90.0% 및 85.7 %, 92.3%, 88.9%로 유의적인 차이를 보이지는 않았다. 본 연구 결과 나타난 BUN 의 수준에 따라서는 발정 유기율 및 발현율에서 유의적인 차이를 보이지 않았지만 BUN 의 수준이 기준치 보다 낮을 경우는 대부분 사료섭취량의 부족한 것으로 판정을 하고 높 을 경우는 사료내 에너지의 부족에 기인한다고 해석을 하는 것이 일반적이다. BCS가 2.25 이하일 때 총 회수된 난자 수는 11.5개로써 2.25 2.75와 2.75 이상일 때 보다 적게 회수됐다. 이식가능 수정란 수에서도 2.5 2.75와 2.75 이상 이상일 때에 비해 2.25 이하 일 때 4.5±0.7개로 5.9±1.8, 5.6±2.3개에 비해 적게 나타났지만 유의적인 차이는 보이지 않았다. 본 연구 결과 BCS 수준별 이식가능 수정란과의 관계에서는 유의적인 차이를 보 이지 않았지만 신체충실지수(BCS)는 소의 영양을 평가하는 방법으로 널리 이용되고 있으 며, BCS의 저하는 번식성적에 나쁜 영향(Markusfeld 등, 1997)을 줄 것이다. 공란우의 과 배란 처리 전․후의 영양상태는 총 회수난자 수 및 이식가능 수정란 수에 영향을 미칠 것 이며, 기전과 요인에 대해서 명확하지 않지만 이러한 영양 수준 및 생화학 수준에 의해 난자 회수율 및 이식가능 수정란 수의 저하가 초래할 것으로 생각된다.

Successful early embryogenesis of somatic cell nuclear transfer (SCNT) embryos is very important to produce cloned animals. However, poor preimplantation development of SCNT embryos has been a major obstacle to the generation of cloned animals due to a lack of understanding of developmental events and underlying mechanism(s). In the current study, we show that production of SCNT embryos with high developmental competence is dependent on the fusion method. Electrofusion causes spontaneous egg activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodeling, whereas Sendai virus (SV)-mediated fusion greatly reduces these events. In addition, SV-SCNT increased the blastocyst development rate and trophectoderm cell number compared to electrofusion-mediated SCNT (E-SCNT). In particular, expression of ER stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos, which could be alleviated by inhibition of ER stress or by using the SV-mediated fusion method. Taken together, these results strongly suggest that SV is a useful fusion material for improvement of preimplantation development of SCNT embryos through reduction of ER stress-associated apoptosis.