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Studies on the In Vitro Development of Cloned Embryos by Somatic Cell Nuclear Transfer in Korean Native Goats (재래산양의 체세포 핵이식에 의한 복제수정란의 체외발달에 관한 연구)

  • Park H. S.;Kim T. S.;Jung S. Y.;Lee Y. H.;Jung J. Y.
    • Journal of Embryo Transfer
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    • v.20 no.2
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    • pp.105-112
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    • 2005
  • The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.

Effects of Donor Cells and Estrus Synchronization on the Production of Cloned Korean Native Goat (공핵 세포 및 발정 동기화가 복제 재래 산양 생산에 미치는 영향)

  • Park H.S.;Kim T.S.;Jung S.Y.;Park J.K.;Lee J.S.;Jung J.Y.
    • Journal of Embryo Transfer
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    • v.21 no.2
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    • pp.137-146
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    • 2006
  • The objective of this study was to examine the effect of donor cell types, the source of recipient oocytes and estrous synchronization on pregnancy and delivery rates of somatic cell nuclear transfer (SCNT) embryos in Korean native goats. Recipient oocytes were surgically collected after superovulation. Ear cells and fetal fibroblasts were collected and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. The zonae pellucidae of in vivo- and in vitro-matured oocytes were partially drilled using a laser system. Single somatic cell was transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3 M mannitol. After the fusion, embryos were activated by Ionomycin+6-DMAP. NT embryos were cultured in mSOF medium supplemented with 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 12 to 20 hr. One hundred and two SCNT embryos were transferred into 20 recipients and pregnancy rate at days 30 was 20.0%. Of them, one developed to term and delivered 1 kid. Ear cells showed significantly higher fusion (63.8 vs. 26.5%) and pregnancy rates (20.0 vs. 0.0%) than those of fetal fibroblast (p<0.05). The recipients synchronized by CIDR showed significantly lower pregnancy rates compared to that of recipient in natural estrus ($0.0{\sim}25.0%$ vs. 100%) (p<0.05). Cloned kid was born from the recipient in natural estrus. For the synchronization of estrus between recipient and donor, there was no difference between treatments (${\pm}0$ vs. +12 hr) in pregnancy rate. The first healthy cloned kid (Jinsoonny) was produced by transfer of SCNT embryos derived from in vivo oocytes and ear cells into a recipient goat whose estrus was synchronized with the donor. These results imply that donor cells for nuclear transfer may affect the success rate, and the estrus synchronization between donor and recipient animals can also be important.

Studies on In Vitro Culture, Freezing and Transfer of Korean Native Cattle Embryos Fertilized In Vitro I. Effect of Co-culture Cells and Growth Factors on In Vitro Development of Korean Native Cattle Embryos Fertilized In Vitro (한우 체외수정란의 체외배양, 동결보존 및 이식에 관한 연구 I. 한우 체외수정란의 체외배양에 대한 공배양세포와 성장인자의 효과)

  • 김일화;손동수;이호준;최선호;양병철;이광원;김경남;장인호
    • Journal of Embryo Transfer
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    • v.11 no.2
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    • pp.111-124
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    • 1996
  • The present study was carried out to investigate the effects of co-culture cells and growth factors on in vitro culture of Korean native cattle(KNC) embryos fertilized in vitro. Two-eight cell embryos were cultured in vitro using 4 types of co-culture cells and 3 growth factors singly or in combination. The results were as follows, In the co-culture of 2~8 cell embryos with bovine oviductal epithelial cell(BOEC), granulosa cell(BGC), uterine epithelial cell(BUEC) and mouse embryonic fibroblast (MEF) monolayers, the developing rate to blastocysts was significantly(P<0.05) higher with BUEC(32.1%) than with MEF(15.3%), BGC(13.2%) and non co-culture control(11.6%). When the morula co-cultured with BOEC for 5 days following in vitro fertilization were co-cultured with BOEC continuously or with BUEC, respectively, the developing rate to blastocysts was higher with BUEC(73.9%) than with BOEC(56.0%). To examine the effects of growth factors on in vitro development of 2~8 cell embryos, epidermal growth factor(EGF), transforming growth factor-$\beta$l(TGF-$\beta$l) and insulin-like growth factor-1(IGF-1) were added singly or in combination to TCM 199 maturation medium with respective concentration. In a addition of each 10, 30 and SOng /rnl EGF, the developing rate to blastocysts was the highest in lOng /ml EGF(25.3%). In addition of each 1, 2 and Sng /mi TGF-$\beta$1, the developing rate to blastocysts was the highest in lng /ml TGF-$\beta$1(28.8%). In addition of each 50, 100ng/ml JGF-l, the developing rate to blastocysts was higher in 100ng/ml IGF-l(16.5%) than in SOng/mi IGF-1(12.9%). When lOng /ml EGF and lng /ml TGF-$\beta$l was added singly or in combination, the developing rate to blastocysts was similar in groups added singly or in combination with EGF and TGF-$\beta$l (23.l~24.6%), although higher than in control(16.7%). In the co-culture of 2~8 cell embryos Wth BOEC + each 10, 30 and 5Ong /rnl EGF, the developing rate to blastocysts was significantly(p<0.05) higher in BOEC + long /ml EGF(32.3%) than in BOEC + 3Ong /ml EGF(18.9%) and BOEC + song /ml EGF(9.7%). In the co-culture of 2~8 cell embryos with BOEC + each 1, 2, Sng /ml TGF-$\beta$l the developing rate to blastocysts was higher in BOEC + Sng/rnl TGF-$\beta$l(28.2%) than in BOEC + lng /ml TGF-$\beta$l(21.7%) and BOEC + 2ng/ml TGF-$\beta$l(21.4%). In summary, higher developing rate to blastocysts were obtained with co-culture of BUEC for co-culture system, with addition of lOng /ml EGF or lng /ml TGF-$\beta$l for growth factor culture system, and with co-culture of BOEC + lOng /ml EGF or BOEC + Sng /ml TGF-$\beta$l for co-culture + growth factor culture system.

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Effect of Fusion Condition on In Vitro Development of Caprine Cloned Oocytes with Nuclear Transfer (재래산양에 있어서 핵이식란의 융합조건이 융합 및 체외발달에 미치는 영향)

  • 박희성;김태숙;이윤희;정수영;이명열;홍승표;박준규;김충희;정장용
    • Reproductive and Developmental Biology
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    • v.28 no.2
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    • pp.127-132
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    • 2004
  • This study was conducted to examine the effects of electric stimulation conditions on in vitro developmental ability of caprine embryos after somatic cell nuclear transfer. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean native goats. The caprine ear cells were cultured in vitro in serum-starvation condition (TCM-l99 + 0.5% FBS) for 3 to 5 days of cell confluence. The zona pellucida of in vivo and in vitro matured oocytes were partially drilled using laser system. Single somatic cell was individually transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol. After the electofusion, embryos were activated by electric stimulation or Ionomycin + 6-DMAP. Nuclear transfer embryos were cultured in mSOF medium supplemented with 0.8% BSA 6∼7 days at 39 , 5% $CO_2$, 5% $O_2$, 90% $N_2$. The fusion rate of donor cells was 60.4% and 40.3 % in ear cell and fetal fibroblast, and cleavage rate were 40.6% and 48.2%, respectively. No significant difference was found in the fusion and cleavage rate in different donor cells. Nuclear transferred oocytes were fused by electric pulses of 1.30∼1.40, 2.30∼2.39 and 2.40∼2.46 ㎸/cm. There was no significant difference among different electric pulses in fusion rates (26.7, 34.8 and 43.8%). The cleavage rate was higher (p<0.05) in 1.30∼1.40 ㎸/cm (82.9%) than 2.30∼2.39 ㎸/cm (43.8%) and 2.40∼2.46 ㎸/cm. (51.8%). The fusion rates of recipient oocyte source were 1st (43.5% and 23.6%), 2nd (55.7% and 39.2%) and 3rd (66.1% and 52.8%) in in vivo and in vitro oocytes. However, fusion ratee were significantly higher (p<0.05) in in vivo than in vitro oocyte. The cleavage rate of fused oocytes from in vivo and in vitro sources were 52.6% and 54.4%, respectively. No significant difference was found in the cleavage rate according to the recipient oocyte source. These results suggest that factors such as field pulse of electric stimulation and oocyte source could affect in vitro developmental ability of nuclear transplanted caprine oocytes.

Effects of Activation Regimens of Recipient Cytoplasm, Culture Condition of Donor Embryos and Size of Blastomeres on Development of Reconstituted Bovine Embryos (수핵 난자의 활성화 방법과 공핵 수정란의 배양체계 및 할구의 크기가 소 핵이식 수정란의 발달에 미치는 영향)

  • 심보웅;조성근;이효종;박충생;최상용
    • Korean Journal of Animal Reproduction
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    • v.22 no.4
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    • pp.425-435
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    • 1998
  • To improve the efficiency of nuclear transplantation in bovine, in this study the development in vitro of nuclear transferred (NT) embryos was compared by different activation regimens of the enucleated oocytes. The effect of developmental stage and culture system of donor nuclei on fusion and development in vitro of NT embryos were also evaluated. Oocytes were collected from Hanwoo ovaries obtained from slaughterhouse and matured in Ham's F-10 supplemented with hormones. After 20~22 h maturation, the oocytes were vortexed to be free from cumulus cells and subsequently their nucleus and the first polar body were removed. Enucleated oocytes were divided into 3 groups for activation; the oocytes of group I were activated with ionomycin for 5 min and subsequently incubated in 6-dimetylarninopurine (DMAP) for 4 h, Those of group II were treated with DMAP for 4 h at 39 h after onset of in vitro maturation (IVM) and those of group III were kept in room temperature ($25^{\circ}C$) for 3 h at 39 h after onset of IVM. After in vitro fertilization (IVF) the embryos for muclear donor were cultured either by group culture (20 embryos /50 ${mu}ell$ drop) or individually (1 embryo /50 ${mu}ell$ drop) for 4 day and 5 day. At day 4 and 5 after IVF, blastomeres were separated in calcium-magnesium free medium, and then classified into small (day 5: $\leq$ 38 ${\mu}{\textrm}{m}$, day 4: $\leq$ 46 ${\mu}{\textrm}{m}$) and large (day 5 : $\geq$ 38 ${\mu}{\textrm}{m}$, day 4 ; $\geq$ 46 ${\mu}{\textrm}{m}$). The separated blastomeres were replaced into enucleated and activated recipient cytoplasm. The blastomere-oocyte complexes were fused by electrically. The NT embryos were cultured in TCM-199 containing 10% FCS in 39$^{\circ}C$, 5% $CO_2$ incubator for 7 day. The results obtained were summarized as follows; There were no differences in fusion and development to blastocyst between groups as group I (68%, 10%), group II (75%, 14%) and group III (73%, 9%), respectively. However, the cell number in blastocyst of NT embryos in group III were significantly fewer than in the other groups (P<0.05). No differences in fusion and development to blastocyst were found between individual or group cultured and between small or large blastomeres of day 4 and day 5 donor embryos. From these results, it was concluded that the combination of ionomycin and DMAP, or treatment of DMAP at 39 h after onset of IVM were useful for the efficient of production of NT bovine embryos, and the individual cultured embryos could be simply used as donor nuclei for NT bovine embryo.

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Effect of OPU (Ovum Pick-Up) Duration on the Rate of Collected Ova and In Vitro Produced Blastocyst Formation (OPU(Ovum Pick-Up) 채란기간이 난자 및 수정란 생산에 미치는 영향)

  • Jin, Jong-In;Kwon, Tae-Hyeon;Choi, Byeong-Hyun;Kim, Sung-Soo;Jo, Hyun-Tae;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.25 no.1
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    • pp.15-20
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    • 2010
  • This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of $3{\sim}4$ days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH and 1 mg/ml of estradiol for 24 h. After 24 h of exposure to sperm, the presumptive zygotes were cultured in CR1aa medium supplemented with 4 mg/ml of BSA for 3 days before being changed to CR1aa medium with 10% of FBS for another $3{\sim}4$ days. The mean numbers of retrieved oocytes were remained constantly up to 3 months ($6.0{\pm}0.5$, $6.2{\pm}0.7$, $5.2{\pm}0.6$), but significantly decreased at over 4 to 6 months ($3.7{\pm}0.5$, $2.8{\pm}0.4$, $1.2{\pm}0.2$) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months ($2.2{\pm}0.3$, $2.5{\pm}0.3$ and $2.3{\pm}0.4$) were significantly higher than those in 4 to 6 months ($0.9{\pm}0.2$, $0.8{\pm}0.2$ and $0.3{\pm}0.2$, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after 3 months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos.

Effect of Early Pregnant Heifer as Donor on the Ovum Pick-Up Derived Oocyte Aspiration and Embryo Production (초기 임신우의 공란우 활용이 초음파 유도 난자 채취 및 수정란 생산에 미치는 영향)

  • Jin, Jong-In;Kwon, Tae-Hyeon;Choi, Byeong-Hyun;Kim, Sung-Soo;Jo, Hyun-Tea;Bang, Jae-Il;Kim, Sam-Chul;Cho, Kyu-Woan;Lee, Jung-Gyu;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.26 no.1
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    • pp.19-25
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    • 2011
  • This study was carried out to evaluate the effect of early pregnant cow as donor for Ovum Pick-Up (OPU) derived oocyte aspiration and embryo production in Holstein heifers. Four non-pregnant and 2 pregnant Holstein heifers were used as donor and then carried out total 17 OPU session for 10 weeks (2 times per week). Recovered cumulus-oocyte-complexes (COCs) were classified into 4 grade by oocyte cytoplasm and cumulus cells and matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol in 5% $CO_2$ and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3 days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes in the early stage pregnant and non-pregnant heifers were $13.0{\pm}4.3$ and $10.6{\pm}3.9$, $5.4{\pm}3.4$ and $7.7{\pm}3.6$ per session, respectively. Rate of collected oocyte from aspirated follicles were 59.2% and 50.5%, respectively. The average number of good quality oocytes (Grade I and II) in the early stage pregnant and non-pregnant heifers was $3.7{\pm}2.7$ and $4.9{\pm}2.6$ (Mean${\pm}$SD). Cleavage and blastocyst developmental rates in Grade I and II were 22.2% and 25.5%, and then $1.7{\pm}0.9$ and $1.4{\pm}1.1$ blastocyst per session, respectively. In conclusion, OPU technology can be used in early stage pregnant and non-pregnant heifers without any problem and so applied OPU derived embryo production to maximize the ability of genetically valuable females.

A Study of The Medical Classics in the '$\bar{A}yurveda$' ('아유르베다'($\bar{A}yurveda$)의 의경(醫經)에 관한 연구)

  • Kim, Ki-Wook;Park, Hyun-Kuk;Seo, Ji-Young
    • Journal of Korean Medical classics
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    • v.20 no.4
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    • pp.91-117
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    • 2007
  • Through a simple study of the medical classics in the '$\bar{A}yurveda$', we have summarized them as follows. 1) Traditional Indian medicine started in the Ganges river area at about 1500 B. C. E. and traces of medical science can be found in the "Rigveda" and "Atharvaveda". 2) The "Charaka" and "$Su\acute{s}hruta$(妙聞集)", ancient texts from India, are not the work of one person, but the result of the work and errors of different doctors and philosophers. Due to the lack of historical records, the time of Charaka or $Su\acute{s}hruta$(妙聞)s' lives are not exactly known. So the completion of the "Charaka" is estimated at 1st${\sim}$2nd century C. E. in northwestern India, and the "$Su\acute{s}hruta$" is estimated to have been completed in 3rd${\sim}$4th century C. E. in central India. Also, the "Charaka" contains details on internal medicine, while the "$Su\acute{s}hruta$" contains more details on surgery by comparison. 3) '$V\bar{a}gbhata$', one of the revered Vriddha Trayi(triad of the ancients, 三醫聖) of the '$\bar{A}yurveda$', lived and worked in about the 7th century and wrote the "$A\d{s}\d{t}\bar{a}nga$ $A\d{s}\d{t}\bar{a}nga$ $h\d{r}daya$ $sa\d{m}hit\bar{a}$ $samhit\bar{a}$(八支集)" and "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$(八心集)", where he tried to compromise and unify the "Charaka" and "$Su\acute{s}hruta$". The "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$" was translated into Tibetan and Arabic at about the 8th${\sim}$9th century, and if we generalize the medicinal plants recorded in each the "Charaka", "$Su\acute{s}hruta$" and the "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$", there are 240, 370, 240 types each. 4) The 'Madhava' focused on one of the subjects of Indian medicine, '$Nid\bar{a}na$' ie meaning "the cause of diseases(病因論)", and in one of the copies found by Bower in 4th century C. E. we can see that it uses prescriptions from the "BuHaLaJi(布哈拉集)", "Charaka", "$Su\acute{s}hruta$". 5) According to the "Charaka", there were 8 branches of ancient medicine in India : treatment of the body(kayacikitsa), special surgery(salakya), removal of alien substances(salyapahartka), treatment of poison or mis-combined medicines(visagaravairodhikaprasamana), the study of ghosts(bhutavidya), pediatrics(kaumarabhrtya), perennial youth and long life(rasayana), and the strengthening of the essence of the body(vajikarana). 6) The '$\bar{A}yurveda$', which originated from ancient experience, was recorded in Sanskrit, which was a theorization of knowledge, and also was written in verses to make memorizing easy, and made medicine the exclusive possession of the Brahmin. The first annotations were 1060 for the "Charaka", 1200 for the "$Su\acute{s}hruta$", 1150 for the "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$", and 1100 for the "$Nid\bar{a}na$", The use of various mineral medicines in the "Charaka" or the use of mercury as internal medicine in the "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$", and the palpation of the pulse for diagnosing in the '$\bar{A}yurveda$' and 'XiZhang(西藏)' medicine are similar to TCM's pulse diagnostics. The coexistence with Arabian 'Unani' medicine, compromise with western medicine and the reactionism trend restored the '$\bar{A}yurveda$' today. 7) The "Charaka" is a book inclined to internal medicine that investigates the origin of human disease which used the dualism of the 'Samkhya', the natural philosophy of the 'Vaisesika' and the logic of the 'Nyaya' in medical theories, and its structure has 16 syllables per line, 2 lines per poem and is recorded in poetry and prose. Also, the "Charaka" can be summarized into the introduction, cause, judgement, body, sensory organs, treatment, pharmaceuticals, and end, and can be seen as a work that strongly reflects the moral code of Brahmin and Aryans. 8) In extracting bloody pus, the "Charaka" introduces a 'sharp tool' bloodletting treatment, while the "$Su\scute{s}hruta$" introduces many surgical methods such as the use of gourd dippers, horns, sucking the blood with leeches. Also the "$Su\acute{s}hruta$" has 19 chapters specializing in ophthalmology, and shows 76 types of eye diseases and their treatments. 9) Since anatomy did not develop in Indian medicine, the inner structure of the human body was not well known. The only exception is 'GuXiangXue(骨相學)' which developed from 'Atharvaveda' times and the "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$". In the "$A\d{s}\d{t}\bar{a}nga$ Sangraha $samhit\bar{a}$"'s 'ShenTiLun(身體論)' there is a thorough listing of the development of a child from pregnancy to birth. The '$\bar{A}yurveda$' is not just an ancient traditional medical system but is being called alternative medicine in the west because of its ability to supplement western medicine and, as its effects are being proved scientifically it is gaining attention worldwide. We would like to say that what we have researched is just a small fragment and a limited view, and would like to correct and supplement any insufficient parts through more research of new records.

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A Study of The Medical Classics in the '$\bar{A}yurveda$' (아유르베다'($\bar{A}yurveda$) 의경(醫經)에 관한 연구)

  • Kim, Kj-Wook;Park, Hyun-Kuk;Seo, Ji-Young
    • The Journal of Dong Guk Oriental Medicine
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    • v.10
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    • pp.119-145
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    • 2008
  • Through a simple study of the medical classics in the '$\bar{A}yurveda$', we have summarized them as follows. 1) Traditional Indian medicine started in the Ganges river area at about 1500 B. C. E. and traces of medical science can be found in the "Rigveda" and "Atharvaveda". 2) The "Charaka(閣羅迦集)" and "$Su\acute{s}hruta$(妙聞集)", ancient texts from India, are not the work of one person, but the result of the work and errors of different doctors and philosophers. Due to the lack of historical records, the time of Charaka(閣羅迦) or $Su\acute{s}hruta$(妙聞)s' lives are not exactly known. So the completion of the "Charaka" is estimated at 1st$\sim$2nd century C. E. in northwestern India, and the "$Su\acute{s}hruta$" is estimated to have been completed in 3rd$\sim$4th century C. E. in central India. Also, the "Charaka" contains details on internal medicine, while the "$Su\acute{s}hruta$" contains more details on surgery by comparison. 3) '$V\bar{a}gbhata$', one of the revered Vriddha Trayi(triad of the ancients, 三醫聖) of the '$\bar{A}yurveda$', lived and worked in about the 7th century and wrote the "$Ast\bar{a}nga$ $Ast\bar{a}nga$ hrdaya $samhit\bar{a}$ $samhit\bar{a}$(八支集) and "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$(八心集)", where he tried to compromise and unify the "Charaka" and "$Su\acute{s}hruta$". The "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$" was translated into Tibetan and Arabic at about the 8th$\sim$9th century, and if we generalize the medicinal plants recorded in each the "Charaka", "$Su\acute{s}hruta$" and the "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$", there are 240, 370, 240 types each. 4) The 'Madhava' focused on one of the subjects of Indian medicine, '$Nid\bar{a}na$' ie meaning "the cause of diseases(病因論)", and in one of the copies found by Bower in 4th century C. E. we can see that it uses prescriptions from the "BuHaLaJi(布唅拉集)", "Charaka", "$Su\acute{s}hruta$". 5) According to the "Charaka", there were 8 branches of ancient medicine in India : treatment of the body(kayacikitsa), special surgery(salakya), removal of alien substances(salyapahartka), treatment of poison or mis-combined medicines(visagaravairodhikaprasamana), the study of ghosts(bhutavidya), pediatrics(kaumarabhrtya), perennial youth and long life(rasayana), and the strengthening of the essence of the body(vajikarana). 6) The '$\bar{A}yurveda$', which originated from ancient experience, was recorded in Sanskrit, which was a theorization of knowledge, and also was written in verses to make memorizing easy, and made medicine the exclusive possession of the Brahmin. The first annotations were 1060 for the "Charaka", 1200 for the "$Su\acute{s}hruta$", 1150 for the "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$", and 1100 for the "$Nid\bar{a}na$". The use of various mineral medicines in the "Charaka" or the use of mercury as internal medicine in the "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$", and the palpation of the pulse for diagnosing in the '$\bar{A}yurveda$' and 'XiZhang(西藏)' medicine are similar to TCM's pulse diagnostics. The coexistence with Arabian 'Unani' medicine, compromise with western medicine and the reactionism trend restored the '$\bar{A}yurveda$' today. 7) The "Charaka" is a book inclined to internal medicine that investigates the origin of human disease which used the dualism of the 'Samkhya', the natural philosophy of the 'Vaisesika' and the logic of the 'Nyaya' in medical theories, and its structure has 16 syllables per line, 2 lines per poem and is recorded in poetry and prose. Also, the "Charaka" can be summarized into the introduction, cause, judgement, body, sensory organs, treatment, pharmaceuticals, and end, and can be seen as a work that strongly reflects the moral code of Brahmin and Aryans. 8) In extracting bloody pus, the "Charaka" introduces a 'sharp tool' bloodletting treatment, while the "$Su\acute{s}hruta$" introduces many surgical methods such as the use of gourd dippers, horns, sucking the blood with leeches. Also the "$Su\acute{s}hruta$" has 19 chapters specializing in ophthalmology, and shows 76 types of eye diseases and their treatments. 9) Since anatomy did not develop in Indian medicine, the inner structure of the human body was not well known. The only exception is 'GuXiangXue(骨相學)' which developed from 'Atharvaveda' times and the "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$". In the "$Ast\bar{a}nga$ Sangraha $samhit\bar{a}$"'s 'ShenTiLun(身體論)' there is a thorough listing of the development of a child from pregnancy to birth. The '$\bar{A}yurveda$' is not just an ancient traditional medical system but is being called alternative medicine in the west because of its ability to supplement western medicine and, as its effects are being proved scientifically it is gaining attention worldwide. We would like to say that what we have researched is just a small fragment and a limited view, and would like to correct and supplement any insufficient parts through more research of new records.

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