• 한국수정란이식학회지
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• 제10권1호
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• 한국수정란이식학회지
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• 제10권1호
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• 한국수정란이식학회지
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• 제11권3호
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• pp.259-269
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• 1996

The present study was undertaken to examine the critical effect of $Ca^2$+ concentration on electrostimulation and post-electrostimulation media for electric activation of in vitro matured oocytes of Korean Native Cattle. Oocytes collected from slaughterhouse ovaries were matured in TCM 199 containing FSH, estradiol-17$\beta$ and FBS with granulosa cell monolayer for 24 hours and denuded with hyaluronidase. And then cumulus-free oocytes were submitted to a DC field of 1.0 kV/cm for 60 $\mu$sec in electroporation media(0.28 M mannito' and PBS) with different $Ca^2$+ concentations (0.00, 0.05, 0.10 and 0.15 mM). Stimulated oocytes were stained and examined for pronuclear formation after incuhation in SOF for 12 hours. The rates of pronuclear formation in hovine oocytes electrically stimulated in 0.28 M mannitol with 0.05, 0.10 and 0.15 mM $Ca^2$+(60.3, 82.2 and 75.0%) were significantly higher than without $Ca^2$+(6.3%) at 12 hours after an electric pulse(p<0.005). The activation rates of Korean Native Cattle oocytes stimulated in PBS supplemented with 0.05, 0.10 and 0.15 mM $Ca^2$+(71.0, 75.8 and 75.4%) were significantly higher than without $Ca^2$+(23.5%) after post-stimulation incubation(p<0.005). After incubation of oocytes in SOF with and without $Ca^2$+ following electric stimulation in 0.28 M mannitol with 0.10 mM $Ca^2$+, the rates of pronuclear formation of bovine oocytes in $Ca^2$+-free SOF(85.7%) was significantly higher than in SOF with 1.71 mM $Ca^2$+(62.5%, p<0.05). When oocytes were stimulated in two electrostimulation media supplemented with $Ca^2$+ and incubated in $Ca^2$+-free SOF, there were no significant differences in the rates of pronuclear formation hetween 0.28 M mannitol and PBS. These results indicate that a single electric pulse could induce activation of Korea Native Cattle oocytes in 0.28 M mannitol and PBS supplemented with $Ca^2$+. Furthermore, to improve the activation rates, it was hetter that stimulated oocytes were incubated in $Ca^2$+-free SOF after electric stimulation than in SOF with $Ca^2$+.

• 한국대기환경학회지
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• 제29권6호
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• pp.818-829
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• 2013

The collection of rainwater samples was made at Jeju area during 2009~2010, and the major ionic species were analyzed. In the comparison of ion balance, conductivity, and acid fraction for the validation of analytical data, the correlation coefficients showed a good linear relationship within the range of 0.966~0.990. The volume-weighted mean pH and electric conductivity were 4.9 and $17.8{\mu}S/cm$, respectively, at the Jeju area. The volume-weighted mean concentrations of ionic species in rainwater were in the order of $Cl^-$ > $Na^+$ > $nss-SO_4{^{2-}}$ > $NH_4{^+}$ > $NO_3{^-}$ > $Mg^{2+}$ > $H^+$ > $nss-Ca^{2+}$ > $HCOO^-$ > $K^+$ > $PO_4{^{3-}}$ > $CH_3COO^-$ > $NO_2{^-}$ > $F^-$ > $HCO_3{^-}$ > $CH_3SO_3{^-}$. The ionic strength of rainwater was $0.26{\pm}0.21$ mM during the study period. The composition ratios of ionic species were such as 50.1% for the marine sources ($Na^+$, $Mg^{2+}$, $Cl^-$), 30.9% for the anthropogenic sources ($NH_4{^+}$, $nss-SO_4{^{2-}}$, $NO_3{^-}$), and 4.7% for the soil source ($nss-Ca^{2+}$), and 3.1% for organic acids ($HCOO^-$, $CH_3COO^-$). From the seasonal comparison, the concentrations of $NO_3{^-}$, $nss-Ca^{2+}$, and $nss-SO_4{^{2-}}$ increased in winter and spring seasons, indicating a reasonable possibility of long range transport from Asia continent. Especially, the acidifying contributions by major inorganic acids ($nss-SO_4{^{2-}}$ and $NO_3{^-}$) and organic acids ($HCOO^-$ and $CH_3COO^-$) were 87.6% and 12.4%, respectively. In comparison by sectional inflow pathway of air mass during the rainy sampling days, the concentrations of $nss-SO_4{^{2-}}$ and $NO_3{^-}$ were relatively high when the air mass was moved from the China continent into Jeju area.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.17-17
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• 2001

착상전 수정란 단계에서 형질전환 수정란의 선발은 형질전환동물의 효율을 증대시킬 수 있는 방법이다. 성공적인 형질전환동물의 생산을 위해서는 생산된 수정란의 mosaicism 빈도를 감소시켜 전체 할구에서의 유전자 발현을 유도하는 것이 최적일 것이다. 따라서 본 연구에서는 돼지의 웅성 생식세포를 이용한 형질전환동물의 생산에 있어서 다양한 정자세포 이용시 형질전환 수정란의 생산성 및 mosaicism 빈도를 조사하였다. 아울러 돼지 웅성생식세포내 GFP 유전자도입시 세포들의 생존율 및 원형정자세포분리 후 배양에 따른 형태적 변화를 관찰하였다. 돼지의 웅성 생식세포내 GFP 유전자 도입은 전기자극법 (1.3 ㎸/cm, 200 $\mu\textrm{s}$) 에 의하여 수행되었으며, 이 때 생존율은 60-70%이였다. 유전자가 도입된 전체 세포중 원형정자세포군의 분리는 유식세포분리기에 의하여 수행하였으며, 전체집단에 대한 분리군의 비율은 평균 16.2%이였다. 형질전환 수정란의 생산은 정자 (ICSI), 원형정자세포 (ROSI), 배양후 확장된 원형정자세포(ELSI)를 이용하였으며 각각의 난할율은 ICSI (82.9%), ROSI (59.1%), ELSI (62.1%)로 유의한 차이를 나타내었다. 그리고 8세포기까지의 배발달율은 각각 61.1, 40.9 및 48.6%이였으며, 상실배 및 포배기형성율은 각각 24.6, 18.1 및 32.4%이였다. 형광현미경하에서 GFP 단백질이 발현된 8세포기 수정란을 대상으로 각각의 할구를 primer extension pream-plification (PEP) PCR 방법으로 분석한 결과, ICSI 및 ROSI 실시후 대부분 (15/20, 9/10) 의 수정란은 3~4개의 할구에서만 GFP 유전자의 존재여부를 확인할 수 있었으며, 전체 할구에서 GFP 유전자가 모두 확인된 수정란은 없었다. 반면에 배양된 확장 원형정자세포를 이용하여 생산한 수정란의 경우, 4/10 (40%)에서 전체 할구내에 GFP 유전자의 존재를 확인할 수 있었다. 이러한 결과는 비록 배발달율 및 GFP 유전자 발현율에 있어서는 ELSI방법이 ICSI 등의 방법보다 현저히 낮았지만, mosaicsism 빈도가 낮아 바람직한 형질전환 수정란 생산에서는 오히려 유용한 방법이라고 사료된다. 또한 외래 유전자의 도입효율 면에서 후기 원형정자나 성숙정자보다 초기 원형정자세포에 외래유전자를 도입한 다음, 성숙시킨 확장원형 정자세포를 이용하는 방법이 보다 우수하다는 것을 시사하였다. 따라서 본 연구결과는 포유동물의 웅성 생식세포를 이용하여 nonmosaicisn을 나타내는 형질전환수정란을 생산하고 선발할 수 있는 일련의 기술적 과정을 정립하였다고 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.74-74
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• 2001

난자 동결방법의 선별은 보다 효과적인 난자은행의 개발에 필수 불가결한 중요한 요소이다. 이전의 연구에서 마우스의 난자를 ethylene glycol과 electron microscope grid를 이용한 유리화 동결법으로 동결 융해한 결과 기존의 slow freezing 방법에서보다 높은 생존율과 배발달율이 나타남을 관찰하였다. 그러나 동결융해후의 난자는 방추사와 염색체의 이상성이 대조군에 비해 높은 빈도로 나타나 융해후의 배발달율을 감소시키는 것으로 보고되었다. 이에 본 연구에서는 유리화동결법동안 항동해제에 Cytoskeleton system을 안정화시키는 cytoskeleton stabilizer인 taxol을 첨가시킨후 동결시켰을때 생존율과 발달율을 개선시킬 수 있는지 알아보고자 본 실험을 시행하였다. ICR mouse의 성숙란을 채취하여 연구목적에 따라 taxol을 첨가시키지 않은 대조군과 첨가시킨 실험군으로 분류하였다. 동결방법은 난자를 1.5 M ethylene glycol (EG)에 2분 30초간 노출시킨후 5.5 M EG와 1 M sucrose가 첨가된 동결액에 20초간 노출시킨 후 Grid에 난자를 부착시킨후 직접 액체질소에 침지하여 동결하였다. 동결후 난자는 5단계로 융해를 실시한 후 정자와 체외수정을 시킨 후 수정된 난자는 modified P1 배약액에 124 h까지 발달율을 관찰하였고, 배양 후 발달된 배반포는 대조군과 실험군, 각각 4마리의 발정동기화된 recipient에 이식을 시행하였다. 배발달율은 대조군에 비해 실험군에서 4세포기 (48 vs. 84.4%), 8세포기 (34% vs. 70.6%), 상실배 (26% vs. 58.6%) 그리고 배반포 발달율은 (24% vs. 58.6%)로 높게 관찰되었다. 배아이식후 대조군과 실험군에서 각각 2 마리가 임신이되어 정상적인 산자를 분만하였다. 따라서 항동해제에 taxol의 첨가는 동결 융해후의 난자의 배발달율을 증진시킬 수 있었다..8%로 나타나 난할율 및 배반포 발생율에 있어서 융합조건에 따라 큰 차이는 없었으나 1.9㎸/cm, 30$\mu\textrm{s}$ 2회의 조건이 다른 조건들에 비하여 유의적으로 낮았다. 따라서, 체세포와 수핵란 세포질간의 융합율과 배반포 발생에 미치는 영향은 전압보다는 시간에 더 크게 받음을 알 수 있었으며, 이와 같은 결과에서 융합시 시간을 오래 주는 것보다 전압을 높이는 것이 수핵난자의 세포질에 상해를 줄이고 이후 배반포 발생에 유리할 것으로 사료되었다.면에서도 더욱 더 활발할 것으로 기대된다. 배란후 72시간째에 초음파진단기를 이용하여 난소의 난포발달을 조사한 결과 , 대조구와 bFF처리구에 비해 AI처리구에서 발달난포가 유의적으로 많은 것을 확인하였다. 이상과 같은 결과로, Anti-inhibin serum은 한우 자체에서 분비하는 Inhibin을 특이하게 억제하여 Inhibin에 의해 억제되는 FSH분비가 촉진됨으로써 난포발달과 estrogen의 농도가 촉진되는 것으로 사료되어 anti-inhibin serum이 한우의 과배란유기 효과가 있는 것으로 사료된다.정량 분석한 결과이다. 시편의 조성은 33.6 at% U, 66.4 at% O의 결과를 얻었다. 산화물 핵연료의 표면 관찰 및 정량 분석 시험시 시편 표면을 전도성 물질로 증착시키지 않고, Silver Paint 에 시편을 접착하는 방법으로도 만족한 시험 결과를 얻을 수 있었다.째, 회복기 중에 일어나는 입자들의 유입은 자기폭풍의 지속시간을 연장시키는 경향을 보이며 큰 자기폭풍일수록 현저했다. 주상에서 관측된 이러한 특성은 서브스톰 확장기 활동이 자기폭풍의 발달과 밀접한 관계가 있음을 시사한다.se that were all low in two aspects, named "the Nonsignificant group". And the issues were high risk perception in general setting and

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.75-75
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• 2001

유리화 동결법은 동결중 ice crystal의 형성이 이루어지지 않으므로 난자의 동결보존을 위한 유용한 동결방법이다. 이전의 연구에서 마우스의 난자를 ethylene glycol과 electron microscope grid를 이용한 유리화 동결법으로 동결 융해한 결과 기존의 slow freezing 방법에서보다 높은 생존율과 배발달율이 나타남을 관찰하였다. 그러나 동물과 인간 난자를 이용한 연구를 통하여 난자의 경우 동결 융해후 염색체에 부착되어 있는 미세소관인 방추사가 온도의 변화에 매우 민감하여 염색체 이상성이 증가되는 것이 보고되었다. 이에 본 연구에서는 성숙난자를 유리화동결법에 의해 동결 융해후 난자의 염색체와 방추사의 이상성이 증가되는지 알아보고자 본 실험을 수행하였다. ICR mouse의 성숙란을 채취하여 연구목적에 따라 fresh한 대조군과 동결음해 시킨 실험군으로 분류하였다. 동결방법은 난자를 1.5 M ethylene glycol (EG)에 2분 30초간 노출 시킨후 5.5 M EG와 1M sucrose가 첨가된 동결액에 20초간 노출시킨 후 Grid에 난자를 부착시킨 후 직접 액체질소에 침지하여 동결하였다. 동결후 난자는 5단계로 융해를 실시한 후 생존된 난자는 tubulin 항체를 이용한 immunostaining 방법으로 방추사의 이상성을 관찰하였고, 염색체는 난자를 고정 후 10% Giemza로 염색 후 염색체의 수적인 이상성을 관찰하였다. 염색체 분석결과 염색체 이상 빈도는 대조군의 경우 19.6%, 동결융해군은 32.8%로 관찰되었다. 또 방추사의 이상빈도는 대조군의 경우 20.2%, 동결 융해군은 32.3%로 관찰되어 동결 융해후의 난자에서 염색체와 방추사의 이상 빈도가 증가됨이 관찰되었다.찰되었다. 배아이식후 대조군과 실험군에서 각각 2 마리가 임신이되어 정상적인 산자를 분만하였다. 따라서 항동해제에 taxol의 첨가는 동결 융해후의 난자의 배발달율을 증진시킬 수 있었다..8%로 나타나 난할율 및 배반포 발생율에 있어서 융합조건에 따라 큰 차이는 없었으나 1.9㎸/cm, 30$\mu\textrm{s}$ 2회의 조건이 다른 조건들에 비하여 유의적으로 낮았다. 따라서, 체세포와 수핵란 세포질간의 융합율과 배반포 발생에 미치는 영향은 전압보다는 시간에 더 크게 받음을 알 수 있었으며, 이와 같은 결과에서 융합시 시간을 오래 주는 것보다 전압을 높이는 것이 수핵난자의 세포질에 상해를 줄이고 이후 배반포 발생에 유리할 것으로 사료되었다.면에서도 더욱 더 활발할 것으로 기대된다. 배란후 72시간째에 초음파진단기를 이용하여 난소의 난포발달을 조사한 결과 , 대조구와 bFF처리구에 비해 AI처리구에서 발달난포가 유의적으로 많은 것을 확인하였다. 이상과 같은 결과로, Anti-inhibin serum은 한우 자체에서 분비하는 Inhibin을 특이하게 억제하여 Inhibin에 의해 억제되는 FSH분비가 촉진됨으로써 난포발달과 estrogen의 농도가 촉진되는 것으로 사료되어 anti-inhibin serum이 한우의 과배란유기 효과가 있는 것으로 사료된다.정량 분석한 결과이다. 시편의 조성은 33.6 at% U, 66.4 at% O의 결과를 얻었다. 산화물 핵연료의 표면 관찰 및 정량 분석 시험시 시편 표면을 전도성 물질로 증착시키지 않고, Silver Paint 에 시편을 접착하는 방법으로도 만족한 시험 결과를 얻을 수 있었다.째, 회복기 중에 일어나는 입자들의 유입은 자기폭풍의 지속시간을 연장시키는 경향을 보이며

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.63-69
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• 2003

Ten barrows, weaned at 28 days (7.2$\pm$0.1 kg BW), were used to evaluate the effects of feeding extruded full-fat soybeans on intestinal morphology and mucosal cell turnover time. All pigs were fed corn-based diets with half of the pigs receiving diets supplemented with 15.5% soybean meal and 3% soybean oil and the remaining pigs fed a diet in which the soybean meal and oil were replaced by 18.5% extruded full-fat soybeans. The pigs were individually placed in $80{\times}150cm$ metabolic cages and fed twice daily an amount approximately equal to their ad libitum intake for a period of 14 days. On day 14, pigs were weighed and then injected intraperitoneally with $^3$H]thymidine ($100{\mu}Ci/kg$ of BW, specific activity 20 Ci/mmol) 6 h after the morning meal. A pig from each treatment was killed 1, 4, 8, 16, or 24 h postinjection and intestinal tissues were collected. Daily gains for pigs fed the soybean diet and extruded full-fat soybean diet were 0.24 and 0.31 kg/day (p=0.05) with feed conversions of 1.58 and 1.39 (p=0.05), respectively. In comparison with pigs fed soybean meal, pigs fed moist extruded full-fat soybeans had a decreased crypt depth in their duodenum and cecum (p<0.1), while the villus height in the mid jejunum and ileum and the total height (villus height plus crypt depth) of the ileum and mid jejunum increased (p<0.05). The villus width in the duodenum and mid jejunum decreased (p<0.05). The number of crypt epithelial cells in the upper jejunum increased but decreased in the ileum, colon and cecum (p<0.05). The number of villus epithelial cells in the ileum and the upper and mid jejunum increased (p<0.05). The time for migration of epithelial cells in the crypt-villus column decreased (p<0.05) in all sites except the upper jejunum, ileum and cecum. The mucosal turnover rate for all intestinal sites except the upper jejunum, colon and cecum decreased (p<0.05). From these data, we conclude that inclusion of moist extruded full-fat soybeans in weanling pig diets can improve the intestinal morphology and slow the migration rate and turnover time of epithelial cells of the small intestine, especially in the mid jejunum compared with soybean meal.

• Journal of Pharmaceutical Investigation
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• 제39권2호
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• pp.97-106
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• 2009

Although lovastatin (LS) is widely used in the treatment of hypercholesterolemia, its bioavailability is known to be around 5%. This study was aimed to increase the solubility and dissolution-permeation rates of LS using solid dispersions (SDs) with bile salts. The solubilities of LS in water, aqueous bile salt solutions and non-aqueous vehicles were determined, and effects of bile salts on the cellulose or duodenal permeation of LS from SDs were evaluated using a horizontal permeation system. SDs were prepared at various ratios of LS to carriers, such as sodium deoxycholate (SDC), sodium glycocholate (SGC) and/or 2-hydroxypropyl-$\beta$-cyclodextrin (HPCD). The addition of bile salts (25 mM) in water increased markedly the solubility of LS by the micellar solubilization. Some non-aqueous vehicles were effective in solubilizing LS. From differential scanning calorimetric studies, it was found that the crystallinity of LS in SDs disappeared, indicating a formation of amorphous state. The SDs showed markedly enhanced dissolution compared with those of their physical mixtures (PMs) and drug alone. In the dissolution-permeation studies using a cellulose membrane, the donor and receptor solutions were maintained as a sink condition using pH 7.0 phosphate buffer containing 0.05% sodium lauryl sulfate (SLS). The flux of LS alone was nearly same as that of LS-SDC-HPCD (1:3:6) PM. However, the flux of LS-SDC-HPCD (1:3:6) SD slightly increased compared with drug alone and PM, suggesting that entrapment of LS in micelles does not significantly hinder the permeation across cellulose membrane. In the dissolution-duodenal permeation studies using a LS-HPCD-SDC (1:3:6) SD, the addition of various bile salts in donor solutions (25 mM) enhanced the permeation of LS markedly, and the fluxes were found to be $0.69{\pm}0.41$, $0.87{\pm}0.51$, $0.84{\pm}0.46$, $0.47{\pm}0.17$ and $0.68{\pm}0.32{\mu}g/cm^2/hr$ for sodium cholate (SC), SDC, SGC, sodium taurodeoxycholate (STDC) and sodium taurocholate (STC), respectively. The stepwise increase of donor SGC concentration increased the flux dose-dependently. From the relationship of donor SGC concentration and flux, the concentration of SGC initiating the permeation across the duodenal mucosa was calculated to be 11.1 mM, which is nearly same as the critical micelle concentration (CMC, 11.6 mM) of SGC. However, with no addition of bile salts and below CMC, the permeation was very limited and irratic, indicating that LS itself is very poor permeable. Higher protions of bile salt in SD such as LS-SDC or LS-SGC (1 : 49 and 1 : 69) showed highly promoted fluxes. In conclusion, SD systems with bile salts, which may form their micelles in intestinal fluids, might be a promising means for providing enhanced dissolution and intestinal permeation of practically insoluble and non-absorbable LS.