The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing β-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a ɤ-tubulin spot. However, ɤ-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-p , EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-p gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

This study was conducted to examine the protein kinase inhibitors, 6-dimethylaminopurine (DMAP) and cycloheximide (CHXM) on the development and chromosome constitution of porcine parthenogenetic embryos. In vitro matured oocytes were activated by electric stimuli (ES) or a combination of ES with culture in 2 mM DMAP or 10 μg/ml CHXM for 4 hr. Activated oocytes were cultured in PZM-3 for 6 days. Some 1-cell embryos and blastocysts were fixed by air dry method to analyze the chromosome constitutions and/or total cell number. Blastocyst development of DMAP-treated group (26.7%) was significantly higher (p<0.05) than those of CHXM-treated and ES control groups. Ploidy in 1-cell stage embryos was not different among groups (77.3 to 81.0%), however, proportion of diploid chromosome constitutions was high in DMAP-treated group (61.9%, p<0.05). In the blastocyst stage, proportion of diploid chromosome plates was significantly high in DMAP-treated group (64.2%, p<0.05), and proportion of abnormal chromosome plates was higher in CHXM-treated group (36.6%, p<0.05) than DMAP-treated group (28.3%,). Proportion of embryos with abnormal chromosome constitutions was slightly increased by DMAP (40.0%) and CHXM (42.1%) treatment due to the increasing of mixoploid (47.4 and 52.0%). The present study shows that the DMAP treatment increase the development of porcine parthenotes. However, parthenogenetic activation by ES or combined treatment with ES and DMAP or CHXM detrimentally affects the chromosome constitutions of porcine parthenotes during early embryonic development, leads to increased abnormal ploidy in the blastocyst stage.

This study was conducted to examine the viability of Korean native striped cattle (Bos namadicus Falconer, Chikso) clone embryos after embryo transfer. Chikso somatic cell nuclear transfer (SCNT) embryos were produced by fusion of ear skin cells derived from a female Chikso with enucleated oocytes matured in vitro for 18-24 hr. After in vitro culture of SCNT embryos for 7 to 8 days, fresh or vitrified blastocysts derived from SCNT were transferred into a uterine horn of recipient cows. Fifteen of total 43 recipients were pregnant at Day 50 and 4 recipients were maintained to term. Three IVF-derived calves and 1 clone Chikso calf were born. Pregnancy rate was higher when fresh embryos were transferred to recipients compared to vitrified embryos, but development to term was not different between both groups. The clone Chikso calf died at 5 days after birth due to the fullness of amniotic fluid in rumen and the infection of umbilical cord. The result of the present study shows that clone Chikso calf can produced from the embryo transfer of SCNT embryos, however, solution of abortion problem is necessary to improve the cloning efficiency.

The aim of this study was to evaluate the effects of orvus es paste(OEP) on the sperm characteristics during freezing in boar semen. Semen quality was evaluated the motility, membrane integrity, mitochondria function, acrosome status, viability and abnormality. Boar semen were frozen until 5℃ for 2 hours using cell freezer and making the straws, and then freezing by lowing the straws into styrofoam box on the 8cm above the LN2 and plunged into LN2 for cryopreservation. In different concentration of OEP (0, 0.25, 0.5 and 1.0%) into cryo-extender, sperm motility, membrane integrity, acrosome status, viability and mitochondria function were significantly higher in 0.5% OEP than those of any other groups, but sperm abnormality were highest in 1.0% OEP group among all treatment groups (P<0.05). In the relationships of the evaluation methods for sperm viability, CBB vs membrane integrity, CBB vs HO/PI and CBB vs mitocondria function were positively correlated (0.67~0.92). Among the evaluation methods, CBB vs membrane integrity, CBB vs HO/PI and CBB vs mitocondria function were significantly correlated (P<0.001). These results of this study indicate that supplementation of 0.5% OEP in boar semen cryo-extender can improve the semen quality.

본 연구는 0.5 ml straw를 이용한 정자 동결융해 시 융해 온도가 동결정자의 성상에 미치는 영향을 파악하고 미니 돼지의 동결정액에 최적화된 적정 융해 조건을 찾기 위하여 실시하였다. 정액동결 straw를 37, 50 및 70℃에서 각각 5초, 10초 및 45초간 융해하여 생존율(SYBR-14/PI staining), 정자원형질막기능검사 (Hypoosmotic Swelling Test) 및 첨체반응율 (CTC : chlorotetracycline staining)을 검사 한 결과 70℃에서 5초간 융해한 정자의 생존율과 CTC 결과가 37℃와 50℃에서 10초 및 45초간 융해한 처리구보다 유의적(p<0.05)으로 높은 생존율과 낮은 비율의 첨체 반응율을 얻었다. 따라서 미니 돼지의 동결 정액은 고온에서 단시간 융해를 하는 것이 정자의 성상에 유리한 것으로 사료된다.

The objective of this study was to establish the optimal conditions for hypoosmotic swelling (HOS) test to assess the functional integrity of the membranes of boar fresh or frozen/thawed spermatozoa. When pooled semen sample was incubated for 30 min at 37℃ with different test solution of varied osmolarity, the highest percentage of HOS positive spermatozoa was observed in a 150 mOsmol fructose/Na-citrate solution (33.6%). Incubation time did not affect significantly the score of HOS positive spermatozoa observed in a 150 mOsmol fructose/Na-citrate solution at 37℃, but the osmolarity affected the score of HOS positive spermatozoa under the same condition above. Fresh semen was significantly better than frozen/thawed semen in semen parameters evaluated such as motility, viability, membrane integrity and lipid peroxidation (p<0.05). In the relationships of sperm parameters, motility vs viability, motility vs membrane integrity and viability vs membrane integrity were positively correlated (0.82～0.94) but lipid peroxidation vs other estimated factors was negatively correlated (－0.90～－0.98). Among the evaluation methods, motility vs viability, motility vs membrane integrity and lipid peroxidation vs other estimated factors were significantly correlated (p<0.05). These results of this study indicate that the optimal condition of HOST in boar spermatozoa is a 150 mOsmol fructose/Na-citrate solution for 30 min incubation at 37℃ and HOST can substitute the examination of motility, viability and lipid peroxidation.

This study was conducted to examine the effect of several saccharides on the induction of capacitation and acrosome reaction (AR) and to examine the effects of mono and polysaccharides on the penetration activity of boar spermatozoa. Spermatozoa were inseminated in medium with fucose, galactose and mannose as monosaccharide, and fucoidan, galactan and mannan as polysaccharide. The penetration rates were significantly (p<0.05) lower in medium with galactose (40.6%), mannose (38.1%), fucose (41.6%) and fucoidan (36.6%) compared with control (56.7%). The rates of AR were increased (40.7 to 59.8%) by the preincubation periods prolonged from 0 to 4 hr (p<0.05). Similar tendencies were observed in AR when spermatozoa were treated with monosaccharides, but not significantly differ among the groups treated with different time of preincubation with some exception of galactose. When spermatozoa were treated with polysaccharides, the rates of AR were significantly (p<0.05) increased by preincubation time prolonged from 0 to 4 hr with an exception of fucoidan. In conclusion, the present study suggests that penetration rate of spermatozoa is higher in presence of polysaccharides than monosaccharides. Also, it may resume that the comparing to control, the all saccharides (L-fucose, D-galactose, D-mannose, fucoidan, galactan and mannan)-treated groups slightly increase the AR pattern as preincubation time prolonged.

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for 30 sec; ES), 2) double electric stimulations (1.5 kV/cm for 30 sec, followed by 1.0 kV/cm for 50 sec after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6～7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

Astaxanthin is a kind of carotenoid compounds, having a antioxidant and anti-inflammatory activities. The antioxidative mechanism by which carotenoid scavenge free radicals has been clearly elucidated, but has not tried for the development of mammalian preimplantation embryo. This study was conducted to investigate the antioxidative effect of astaxanthin on in vitro development of porcine in vitro fertilized embryos. Porcine embryos derived from in vitro fertilization (IVF) were cultured in 5% CO2 in air at 38.5℃ in PZM-3 medium supplemented with different dosages of astaxanthin (0, 1, 5 and 10 M) and taurine (0, 1, 2.5 and 5 mM) as a positive control, and execute to compare the effects of various antioxidants such as taurine, melatonin and asculatin on in vitro development. The proportions of embryos developed to the blastocyst stage were increased when 1 and 5 M of astaxanthin (26.6 and 23.4%, respectively) and 1 and 2.5 mM taurine (25.8 and 26.4%, respectively) were supplemented, compared to controls (p<0.05). Also, various antioxidant-treated groups were significantly higher rates of blastocysts (astaxanthin, 27.4%; taurine, 29.1%; melatonin, 26.8%; aesculetin, 27.9%, respectively ) than control (18.8%). There was no difference in mean cell number of blastocysts between antioxidants and control. This result indicates that astaxanthin has an antioxidant feature when porcine IVF embryos were cultured in vitro.