This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or 10 μM Ca2+-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in 25 μs than 20 and 30 μs (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann’s (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann’s cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by 5 μM Ca2+-ionophore was significantly higher than the rates of the embryos activated with 10 μM of Ca2+-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in 10 μM Ca2+-ionophore than 5 μM counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for 25 μs in Zimmermann’s fusion buffer followed by artificial activation with 5 μM Ca2+-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.

A total of 222 udder-half milk samples of lactating goats were collected from two herds in Korea during 2008 and all samples were subjected to bacteriological examination. Somatic cell counts (SCC) were also determined for all samples except for 13 (5.9%), which were collected from halves of udders with clinical mastitis. A total of 85 bacteria were isolated from 82 (36.9%) of 222 milk samples tested. Staphylococci were the predominant pathogens, accounting for almost 70% of the isolates: Coagulase negative staphylococci (CNS) and S. aureus constituted 55% (47/85) and 14.1% (12/85), respectively. Among 209 samples tested for SCC, bacteria were isolated from 36 of 115 (31.3%) samples with SCC of <1×106 cells/㎖ and 38 of 94 (40.4%) samples that had SCC of ≥1×106 cells/㎖, respectively. All S. aureus were detected from samples with SCC of ≥1×106 cells/㎖, while 25 of 47 (61.0%) CNS were isolated from milk samples with SCC of <1×106 cells/㎖. Mean SCC of milk samples that harbored S. aureus and CNS was 4,787×103 cells/㎖ and >1×106 cells/㎖, respectively. All S. aureus and CNS isolates were susceptible to all antimicrobials tested except for penicillin, to which 2 (16.6%) S. aureus and 12 (25.5%) CNS isolates showed resistance.

Somatic cell nuclear transfer (SCNT) is a useful tool for reproducing genetically identical animals or producing transgenic animals. Many reports have demonstrated that the efficiency of animal cloning by SCNT requires reprogramming of the somatic nucleus to a totipotent like-state. The SCNT-related reprogramming might mimic the natural reprogramming process that occurs during normal mammalian development. However, recent evidence indicates that the reprogramming event by SCNT is incomplete. In this study, the traditional SCNT procedure (TNT) was modified by injecting donor nuclei into recipient cytoplasm prior to the enucleation process to expose the donor nucleus before removing the karyoplast containing the chromosomes of the oocytes which might possess additional reprogramming factors, and this modified technique was named as reversing the usual order of SCNT (RONT). Other procedures including activation and in vitro culture were the same as TNT. Contrary to expectations, the rate of blastocyst development was not different significantly between RONT and TNT (8.6% and 7.9%, respectively). However, duration of micromanipulation performed by the same technician and equipments was remarkably reduced because the ruptured oocytes after nuclear injection were excluded from the enucleation process. This study suggests that RONT, a simplified SCNT protocol, shortens the duration of SCNT procedure and this less time-costing protocol may enable the researchers to perform murine SCNT easier.

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 (≥ 2-cell) embryos were cultured in 0 (control), 1, 10 and 20 μM flavonoid for 6 days. In the results, in vitro development rate was the highest in 10 μM flavonoid group (57.1%) among treatment groups (control, 49.5%; 1 μM, 54.2%; 20 μM, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in 10 μM flavonoid group than other groups. We found that 10 μM flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in 10 μM flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, 10 μM flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in 10 μM flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing γ‐gtubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The γ‐gtubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas γ‐g tubulin spots in the SCNT embryos were observed only during the inter‐ganaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum‐starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or 150 μM roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine‐treated groups (27.6%) than that of the roscovitine‐treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine‐treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine‐treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine‐treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine‐treatment creates a more suitable condition for nuclear reprogramming after SCNT.

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.