Earticle

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD46 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace × Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm,, 75.4%), the fusion rate of the GalT/CD46 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less than 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted (30×106 spermatozoa/ml) in different extenders. Semen was stored at 17℃ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection. The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significantly different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.

Although the function and utility of RNA transcripts derived from matured spermatozoa remains unclear, they might play important roles in the establishment of a paternal genome and subsequently embryo development. Herein, we investigated the expression of X-chromosome linked RNA transcripts in matured bovine spermatozoa. The total RNA was extracted from the matured spermatozoa, and then converted to cDNA. Autosomal genes (ACT-β and H-2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) were analyzed for the characterization of X-chromosome linked RNA transcripts and compared to female fibroblasts by RT-PCR. The transcripts of autosomal genes (ACT-β and H2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X and ZFX) were not detected in spermatozoa. However, XIAP (X-linked inhibitor of apoptosis protein) and XIST (X-chromosome inactive-specific transcript, a kind of paternal imprinted gene) transcripts were detected in spermatozoa, and relative levels of XIAP and XIST transcripts were similar and 0.5-fold lower when compared to female fibroblasts, respectively. Based on the findings, it is summarized that the presence of RNA transcripts of XIAP and XIST in the isolated spermatozoa may imply their role in inhibition of apoptosis and induction of X-chromosome inactivation in embryo development.

Acyl-CoA synthetase 4(ACSL4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. The human and mouse ACSL4 gene are mapped on chromosome X. The female mice heterozygous for ACSL4 deficiency became pregnant less frequently and produced small litters, with 40% of embryos surviving gestation. In this study, we examined the regulation of ACS4 by estradiol-17β and progesterone (P4) in the human endometrial cancer cell line HTB-1B. ACSL4 mRNA was increased in a dose-dependent manner. Also, expression of ACSL4 gene was up-regulated in a time-dependent manner in HTB-1B cells. However, combined treatment with progesterone and estradiol-17β modestly decreased the levels of ACS4L mRNA as compared with the estradiol-17β and progesterone respectively. Overall, these results suggest that the ACSL4 gene is regulated by progesterone and estradiol-17β in the HTB-1B cells.

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide (H2O2), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decreased with addition of H2O2, and were significantly (p<0.05) lower in medium with 0.1 mM H2O2 than control group. Also, the rate of degenerated oocytes was increased in as H2O2 concentration increased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When H2O2 concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM H2O2 and (3) control medium with 1.0 mM H2O2 along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. H2O2 decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity against oxidative stress caused by H2O2. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM H2O2 alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

This survey was conducted to investigate the current status of swine artificial insemination(AI) centers registered as 'semen processing business' in Korea. The survey responses were collected by direct visitation or telephone conversation for 5 months from May through September in 2008. The survey showed that sixty-four AI centers were enrolled in local government and those of fifty-two AI centers were under operation. Forty-nine AI centers surveyed owned a total of 3,334 boars and the Duroc breed accounted for the highest rate(73.1%) of all boar breeds. In type of ownership, agricultural management corporations was the highest(42.3%) and followed by private ownership (34.6%). Large-scale AI centers in terms of own over 151 boar were surveyed as 5.9% and most AI centers own less than 100 boars(86.5%). The average number of boars per AI center was 68. The amount of liquid semen provided by 52 AI centers were 1,791,000 doses and each AI center provides average of 39,000 does, which is represented for 90% consumption by sows in Korea.

This study was performed to investigate the FSH levels for superovulation procedure in Korean Native Cattle (Hanwoo). The effectiveness of 200 mg and 400 mg of FSH to initiate superovulation was examined in Hanwoo. Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH group was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. Also, 400 mg FSH group was treated with 80, 60, 40, 20 mg FSH levels. On the 3rd day administration of FSH, 25 mg PGF2α was administered and CIDR was withdrawn. Donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 μg GnRH at time of 1st insemination and embryos were recovered 8 days after the 1st insemination. As a results, average number of CL treated with FSH 200 mg was higher as 20.9±1.20 than 15.8±0.63 for donors treated with FSH 400 mg, respectively(p<0.05). Treated group of 200 mg FSH level increased (p<0.05) the number of embryos recovered per procedure compared to 400 mg FSH level (18.2±1.18 vs. 12.38±0.52, respectively). When treatment of 200 mg FSH was performed, average transferable embryos/ova increased (p<0.05) to 14.1±1.12 from 6.8±0.33 of treated of 400 mg FSH. Group of 200 mg FSH increased (p<0.05) to 8.3±0.76 from 2.0±0.26 in morula stage compare to 400 mg FSH group. Mean of total early blastocyst and expanded blastocyst stage embryos was similar (p<0.05) between the 200 mg and 400 mg FSH levels group (4.7±1.19 vs. 2.9±0.18 and 1.2±0.40 vs. 1.9±0.17). These results suggest that 200 mg FSH level-based superovulation protocol with CIDR may be effectively used for production of superior embryos in Hanwoo. In other words, the less level of FSH may be effectively applied for Hanwoo (Korean Native Cattle), because Hanwoo was smaller body size than beef or daily cow.

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These facts suggest that expression vector system is normally expressed in the trnasgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.

The objective of this study was to find out candidate genes associated with litter size trait in pigs of inbred Large Yorkshire and Landrace populations. 86 sows were screened for candidate genotypes along with litter size data recordings. Association of litter size with genotypes of candidate genes were investigated to verify the usefulness of each gene's genotypes as markers for the trait. For the lines of Large Yorkshire, PRLR3 and RBP4 genes were genotyped. Frequency distribution of PRLR3 with genotypes AA, AB and BB were each 0.14, 0.44 and 0.42. And the average litter size by PRLR3 genotypes were 8.83, 10.81 and 10.70 piglets per litter, the average estimated breeding values of which were 0.243, 0.332, 0.365, respectively for AA, AB and BB genotypes. Genotypic frequencies of RBP4 by AA, AB and BB genotypes were 0.10, 0.44 and 0.46. The average litter size by genotypes of RBP4 were 10.40, 10.57 and 10.35 piglets per litter and their corresponding average estimated breeding values were 0.451, 0.353 and 0.261, respectively for genotypes AA, AB and BB. Significance in differences among genotypes were not observed, but B allele of RBP4 seems to be associated with litter size. In Landrace lines, frequencies of RBP4 genotypes, AA, AB and BB were 0.29, 0.55 and 0.16. And the average litter size of these genotypes were 10.50, 11.08 and 11.00 piglets per litter. The corresponding averages of estimated breeding values of each genotypes were 0.172, 0.135 and 0.104. In Landrace lines, allele A was more likely to be associated with litter size, even if differences among average litter size were not significant. We conclude that genotyping of two candidate genes is a helpful tool to identify genetic potentials of litter size in pigs.

Human growth hormone (hGH), one of the most important hormones in medicine, is secreted from anterior pituitary gland. Its broad physiological function includes body growth, cell regeneration, increasement of muscle volume, bone density, body fat reduction, and so on. Due to the wide range of therapeutic effects, the hGH produced from E. coli has been commercialized already. In this study, we asked whether it is possible to produce recombinant hGH efficiently from various cultured mammalian cells. To meet this purpose, we chose a retrovirus vector system for transfer and expression of the hGH gene in various mammalian cells. Analyses of RT-PCR, ELISA, and Western blot to determine expression of the hGH gene showed the highest production of the hGH was determined from chicken embronic fibroblast (CEF) cells with the concentration of 8.58 μg/ml. The biological activity of the hGH was similar to the commercially available counterpart. These results suggest that mass production of hGH is possible not only in the E. coli but also in the various mammalian cells.

In general, the blood cell culture is a common method for animal chromosome preparation. However, every animal and its cells have unique physiological characteristics and functions. Hence, it is very difficult to find the suitable method of chromosome preparation using animal lymphocyte culture. This study was carried out to find the suitable method of chromosome preparation using lymphocytes cultures in mammalians and aves including cattle, rat, mouse and chicken. To seek the optimal method of lymphocyte culture in each animal, 23 factorial experiment was designed. The design evaluated three main effects in culture duration, kinds of mitogen supplements and colcemid exposure time with two levels within each effect. The mitotic index and the score of chromosome morphology were analyzed. In results, the suitable methods of lymphocyte culture for chromosome preparation were 72 hours culture, pokeweed mitogen(PWM) supplement and 90 minutes of colcemid exposure in cattle, 72 hours culture, PWM supplement and 50 minutes of colcemid exposure in chicken, 96 hours culture, concanavalin A supplement and 90 minutes of colcemid exposure in rat, and 72 hours culture, PWM supplement and 50 minutes of colcemid exposure in mouse, respectively. In conclusion, kinds of mitogen, culture duration and colcemid exposure time significantly affected the mitotic index and chromosome morphology in animal lymphocyte culture. The interaction effects between/among treatment factors were also statistically significant.

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until 5℃ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time. sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

It was conducted the experiment, divided into three groups as normal, poor and polycystic ovary syndrome, to detect the change of protein patterns in follicular fluid on ovarian response following controlled ovarian hyperstimulation for human IVF outcome. In the normal group, it was confirmed reproducible 57 spots in the detected total 81 spots. Then 1 spot was not found in the other groups. In the poor responder group, it was found reproducible 53 spots in the detected total 98 spots. 6 spots were down-regulation and 7 spots were up-regulation comparable with normal group. There were not 5 spots in poor responder group comparable with other groups. In the polycystic ovary syndrome group, it was expressed reproducible 53 spots in the detected total 80 spots and 3 spots were just expressed in this group. However, 4 spots were not found in polycystic ovary syndrome. 9 spots were up-regulation comparable with normal group. Significant up and down-regulation spots among the each groups were identified. The results were a cytosolic carboxypeptidase, a signal-induced proliferation-associated protein 1, a ceruloplasmin, a keratin(type Ⅱ cytoskeletal 1), a polypeptide N-acetylgalactosaminyltransferase 2, a serine/threonine-protein phosphatase 4 regulatory subunit 4. It was identified that 8 spots, 6 kinds of protein are corresponded with NCBInr database research, but 10 spots were failed in the identification. In conclusion, it has been confirmed change and expression of protein on the ovarian response following COH of human.

In the present study, we identified differentially methylated region (DMR) upstream of Dnmt1o and Dnmt1s gene in early porcine embryos. Porcine Dnmt1o had at least one DMR which was located between —530 bp to —30 bp upstream from transcription start site of the Dnmt1o gene. DNA methylation analyses of Dnmt1o revealed the DMR to be hypomethylated in oocytes, whereas it was highly methylated in sperm. Moreover, the DMR upstream of Dnmt1o was gradually hypermethylated from oocytes to two cells and dramatically changed in the methylation pattern from four cells to BL stages in an in vivo. In an IVF, the methylation status in the DMR upstream of Dnmt1o was hypermethylated from one cell to eight cells, but demethylated at the Morula and BL stages, indicating that the DNA methylation pattern in the Dnmt1o upstream ultimately changed from stage to stage before the implantation. Next, to elucidate whether DNA methylation status of Dnmt1s upstream is stage-by-stage changed in during porcine early development, we analyzed the dynamics of the DNA methylation status of the Dnmt1s locus in germ cell, or one cell to BL cells. The Dnmt1s upstream was highly methylated in one and eight cells, while less methylated in two, four, morula, and BL cells. Taken together, our data demonstrated that DNA methylation and demethylation events in upstream of Dnmt1o/Dnmt1s during early porcine embryos dramatically occurred, and this change may contribute to the maintenance of genomewide DNA methylation in early embryonic development.

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

The objective of this study was to evaluate efficiency of methyl-beta-cyclodextrin (MBCD) in the sperm preservation of bull. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 1, 5, 10 and 20 mM MBCD before freezing process. Analysis of viability in frozen-thawed sperm was estimated by SYBR14/PI double stain, hypoosmotic swelling test(HOST) and acrosome damage with FITC-PNA, and mitochondria activation with Rhodamin123 by flow-cytometry. The sperm viability was significantly higher in 0 mM and 5 mM concentrations of MBCD than other groups (p<0.05). However, the HOST was significantly lower at 20 mM concentration of MBCD than other concentrations (p<0.05). In addition, acrosome damage and mitochondria activation rates were significantly lower at 20 mM concentration of MBCD than other groups (p<0.05). In conclusion, the viability of sperm was not significantly different among concentrations of MBCD 0, 5 and 10 mM, but MBCD 20 mM was significantly lower than other groups. In addition, as concentrations of MBCD was high, HOST, acrosome damage and mitochondria activation rates had a negative effect in bull sperm.

Fibroblasts of large animals are easy to isolate and to maintain in vitro culture. Thus, these cells are extensively applied to donor cell for somatic cell nuclear transfer, and to substrate cells to generate induced pluripotent stem cells after transfection of required genes to be essentially required for direct reprogramming. However, limited mitotic activity of fibroblasts to differentiate along a terminal lineage becomes restrictive for their versatile application. Recently, commercial culture medium and systems developed for primary cells are provided by manufactures. In this study, we examined whether one of the systems developed for primary fibroblasts of human are effective on porcine ear skin fibroblasts. To this end, we performed proliferation assay after five days culture in vitro of porcine fibroblasts in medium DMEM, which is generally used for fibroblasts culture, and medium M106 for human dermal fibroblasts, supplemented with various concentrations of FBS and LSGS contained mainly growth factors, respectively. Consequence was that presence of 15% FBS and 0.1 X concentrations of LSGS in DMEM showed most active proliferation of porcine fibroblasts.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG (33.1±9.6 vs 21.7± 8.3%). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP- treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The m-RNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl2 in Ca-free CZB medium in the presence of 5 μg/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6～7 week-old mice(53.3%) than in oocytes from 8～9(80.9%) and 10～11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium (51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6～7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM CaCl2 ․ 2H2O), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM CaCl2 ․ 2H2O) supplemented with 100, 200 or 300 μg/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 μsec. The activated embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 μg/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM CaCl2 ․ 2H2O (T1), 1.0 mM CaCl2 ․ 2H2O (T2) and 0.1 mM CaCl2 ․ 2H2O with 100 μg/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-α/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen ejaculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution Ⅰ: 11% lactose+egg yolk; solution Ⅱ: solutionⅠ+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until 5℃ for 1 hrs, holding at —102℃ for 10 min; B: until 5℃ for 2 hrs, holding at —102℃ for 10 min; C: until 5℃ for 3 hrs, holding at —80 and —102℃ for 10 min). Semen cooled until 5℃ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1～2 hrs, 1～3% glycerol concentrations and glycerol equilibrium time of 0～10 min.

The objective of this study was to investigate the relationship between concentration of urea nitrogen, glucose, cholesterol and number of transferable embryos for the purpose of improving reproductive performance in blood of Hanwoo donors. Fifty five, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg PGF2α was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100μg GnRH at the time of 1nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. Cows with BUN ＜10, 11～18 and ≥19 mg/dl had number of transferable embryos of 4.3±1.3, 5.8±1.8 and 4.7±2.1 respectively. The mean numbers of total ova from < 10 and 10≤ of corpora lutea(CL) was 8.9 and 14.3, respectively. The number of transferable embryos differed between < 10 and 10≤ CL was 4.8 and 5.6, respectively.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

The objective of this study was to investigate the relationship between estrous expression, body condition score (BCS), blood urea nitrogen (BUN) and number of transferable embryos for the purpose of improving reproductive performance in blood of Hanwoo donors. Sixty, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg PGF2α was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100 μg GnRH at the time of 1nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. The estrous inducement rate and estrous expression rate were significantly lower for cows with BCS below 2.25 than for cows with BCS above 2.25. There was 50.0% of rate of mounting in cows with BCS below 2.25 whereas the rate of mounting was markedly increased in cows with BCS above 2.25 (94.1% and 89.5% for BCS 2.25～2.75 and BCS above 2.75 cows, respectively). Cows with BCS ＜2.25, 2.25～2.75 and ≥2.75 had number of transferable embryos of 4.5±0.7, 5.9±1.8 and 5.6±2.3 respectively.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl® solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for 5 min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

The aim of this study was to evaluate the effect of α-tocopherol on blastocysts development and subsequent cryosurvival of the vitrification. The α-tocopherol(0, 100, 200, 400 μM) was added in to culture medium for the bovine embryos. The blasocysts from the α-tocopherol and untreated control groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. There were no differences in the overall cleavage rate(56.14±4.66, 58.18±4.70, 62.97±6.86 and 51.17±7.28) among four treatment groups. However, in blastocyst development and total cell number were significantly higher in α-tocopherol 200 μM(38.60±7.12; 106.33±3.50) to culture medium than other treatment groups(29.30±5.24, 31.60±7.12 and 26.37±4.18; 101.36±5.12, 97.27±2.87, and 91.23±7.52 respectively). Before and after vitrification, the total cell number and blastocyst development of embryo were significantly higher in July to August than September to October. In conclusion, addition of α-tocopherol 200 μM to in vitro bovine embryo culture medium was beneficial for improving embryo quality by decreasing the embryo damage blsstocysts cell number and improving the tolerance of the embryos to cryopreservation.

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into LN2. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol (72.5±5.00%, 54.88±0.66% and 46.00±2.40%; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol (34.69±4.64% vs 46.00±2.40%; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: 55.81±2.94, 55.19±3.34 vs 47.94±3.48%; p<0.05 and membrane integrity: 44.94±3.51, 46.06±2.25 vs 40.38±1.03%; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

This study was carried out to investigate the effects of semen on reproductive ability in crossbred Korean native chicken (KNC, 58-wk old). The body weight, volume of semen and concentration of spermatozoa, were 2.96 g, 0.40 ml, 36.58×108/ml, respectively, in KNC. The fertility and hatchability were 94.8% and 78.8% respectively in crossbred KNC. KNC(Y) was high compared to other strains in fertility. The other strains were not significantly different among 6 strains. The results of this experiment indicated that hatchability of (G) was high compared to other strains. The result of this study could be available to genetic improvement of reproductive traits as a basic reference in KNC strains. To achieve the more effective improvement of reproductive traits, addition research such as genetic parameter evaluation should be performed.