This study was undertaken to access the effects of collection method, room temperature at oocyte recovery and culture media on the oocyte quality, fertilization and cleavage rates of in vitro matured and fertilized oocytes of Korean native goats. Ovaries obtained from a slaughterhouse were transported to the laboratory and were divided into 2 groups. One group of ovaries was maintained at 30 to 35$^{\circ}C$ of the room temperature and another group was remained at 20 to $25^{\circ}C$ during oocyte recovery. The oocytes were recovered by follicle aspiration, slicing and aspiration+slicing methods from 3 groups of follicles according to size; <2 mm, 2 to 6 mm and >6 mm. The matured oocytes were inseminated with buck epididymal spermatozoa at a concentration of 3~3.5$\times$10$^{6}$ m1 and fertilization was identified when 2 pronuclei were present in the cytoplasm. Although the recovery rate per ovary obtained by the combination of follicle aspiration + slicing(19.6$\pm$2.2) method was higher than aspiration(11.7$\pm$1.1) and slicing(14.8$\pm$1.8) collection, optimal recovery according to oocyte grades resulted form ovarian slicing compared to aspiration or combined methods(P<0.05). However, no significant differences were found in the mean number(2.5$\pm$1.8; 3.3$\pm$3.3; 2.9$\pm$2.4) and the proportion of favorable oocytes(Grades I, II and III) recovered(31.6%, 36.0%, 36.4%,) according to follicle size(<2 mm; 2 to 6 mm; >6 mm). Fertilization rate was 60.0%, 67.7%, 70.6% and 56.4% and the proportion of embryos/zygotes was 11.1%, 7.1%, 5.0% and 2.8% in 20~$25^{\circ}C$/BO, 30~35$^{\circ}C$/BO, 20~$25^{\circ}C$/TALP and 30~35$^{\circ}C$ /groups, respectively.
This study was carried out to investigate the factors affecting maturation in vitro of follicular oocytes in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro in TCM-199 containing FCS and hormones. The effects of TCM-199 salt type, number of oocytes per drop, incubation time and co-culture with granulosa cells on maturation of oocytes, were investigated. The results obtained are summarized as follows. 1. The maturation rates of follicular oocytes cultured for 22, 25 and 28 hours in Hank's TCM-199 or Earle's TCM-199 were 59.3, 59.6, 80% and 80.0, 90.0, 93.7%, respectively. The maturation rate of follicular oocytes in Earle's TCM-199 was faster and higher than in Hank's TCM-199(P<0.05). 2. The maturation rates of oocytes were 54.5, 62.5 and 62.0% when cultured the oocytes number 10, 20 and 40 per 200${mu}ell$ drop for 18 hours. 3. The maturation of follicular oocytes in the Korean Native Cattle was induced at 14 hours culture, by giving the maturation rate of 90.0% after 20 hours. 4. The maturation rates were 63.3% and 66.7%, respectigely when the oocytes were co-cultured with granulosa cells from medium-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$). When the oocytes were co-cultured with granulosa cells from medium-size follicles cultured for 3 days, the maturation rates were 20.8% and 76.2%, respectively(P<0.05). 5. The maturation rate were 88.0% and 70.0%, respectively when the oocytes were co-cultured with granulosa cells from large-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the oocytes were co-cultred with granulosa cells from large-size follicles cultured for 3 days, the maturation rates were 41.2% and 73.3%, respectively(P<0.05).
Park, Chung-Saeng;Jo, Seong-Geun;Lee, Jeong-Gyu;Gang, Tae-Yeong;Park, Seong-Jae;Gong, Il-Geun;Choe, Min-Cheol
Korean Journal of Animal Reproduction
/
v.21
no.2
/
pp.147-156
/
1997
Ultrasound-guided follicular aspiration was performed in Holstein heifers once weekly with or without pretreatment of single or multiple decreasing doses using a total of 400 mg FSH. Oocytes were aspirated with a 6.5 MHz convex-array ultrasound trasducer designed for intravaginal use. All the visible follicles larger than 4 mm in diameter were punctured with a 17 gauge, 55 cm needle at each aspiration session and the follicular fluids containing oocytes were obtained by vacuum suction. The results obtained were as follows: As a preliminary experiment, the recovery rates of folicular oocytes by ultrasound-guided aspiration from the isolated ovaries of Korean native cows were compared between suction methods using manual syringe or vacuum pump. The recovery rate of oocytes using vacuum pump (80.7%) was significantly (P<0.05) higher than that using manual syringe (47.1%). The follicles were counted by their size in diameter with ultrasound image, and recovery rates and grades of follicular oocytes collected by ultrasound-guided aspiration were investigated in Holstein heifers pretreated with or without FSH. A group of heifiers were injected with multiple decreasing doses (twice a day for 3 days) of a total of 400 mg FSH. The other 2 groups were injected with a single dose of 400 mg FSH mixed with 25% PVP. Ultrasound observation of follicle population and/or ultrasound-guided transvaginal oocyte aspiration were performed 12 hrs following the last FSH injection in the multiple dose group, and 48 or 60 hrs after FSH injection in the single dose groups. Most of the visible follicles had small size of less than 3 mm in diameter in unstimulated heifers (71.0%), but medium size in all the heifers treated with FSH. (70.5 to 92.8%). The number of OPU follicles per session (4.6$\pm$1.9) were much less, compared to the vilsible follicle counts (9.7$\pm$2.2), in the nustimulated heifers due to the small dominant follicles. Among 4 goups of heifers the most visible as well as OPU follicles were observed in the heifers at 60 hrs following treatment of a single dose of 400 mg FSH (21.2$\pm$2.3 and 21.0$\pm$2.0), and the differences in both the follicle counts between the groups was found significant (P<0.05) The rates of oocyte recovery from the follicles by ultrasound-guilded aspiration were varied 46.3 to 75.0% in the heifers unstimulated and treated with a single dose of 400 mg FSH, but the group difference was not significant. The number of recovered oocytes per session a, pp.ared to be highest at aspiration at 60 hrs following single FSH (10.6$\pm$2.2) than at aspiration at 48 hrs after single FSH (7.8$\pm$2.7) or in the unstimulated heifers (3.4$\pm$3.0). The proportion of grade I and II oocytes to all oocytes collected was varied 31.8 to 64.0% between the groups. However, there was found no significant difference in both the number of oocytes recovered per session and the percentage and the percentage of grade I and II oocytes. From the above results it was concluded that the more oocytes of superior quality might be recovered economically by ultrasound-guided aspiration at 60 hrs following the pretreatment of a single dose of 400 mg FSH and by suction using a vacuum pump system of about negative pressure of 75 to 85 mmHg.
Park, H.-S.;Lee, Y.-H.;Kim, T.-S.;Park, J.-K.;Lee, J.-S.;Kim, C.-H.;Jung, J.-Y.
Journal of Embryo Transfer
/
v.19
no.2
/
pp.81-87
/
2004
This study was designed to determine whether repeated superovulation is beneficial for recovery and quality of oocytes in Korean native goats. Seventy-six mature goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen impregnated CIDR for 10 days and then the goats were divided into two groups. One group of the goats received a single intramuscular injection of 1,000 IU PMSG on Day 8 of CIDR insertion. The other group of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF2{\alpha}on$ Day 8 and 400 IU hCG in the afternoon on Day 10. For oocyte recovery, donor goats were fasted 24 h before operation. Anesthesia was induced by intravenous injection of 2% xylazine(0.2 mg/kg body weight) and ketamin(11 mg/kg body weight). In vivo oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 hours after hCG injection through mid-ventral incision. The mean number of CL and oocytes recovered and recovery rate of oocytes by oviduct flushing were greater(P<0.05) in the first treatment than those in the second treatment. Contrary to our assumption, PMSG treatment significantly (P<0.05) increased the number of CL formed and recovery rate of oocytes compared to FSH. However, the same effect was not observed in recovery of follicular oocytes. There was no significant difference in oocyte quality between FSH and PMSG or first and second treatments. The present results indicate that repeated superovulation and repeated use of donor animals may be inefficient for obtaining oocytes in good qualities.
In viro fertilizatin is very important in both human clinical practice and animal breeding. However, the success rate of in vitro fertilization is not high. The purpose of this study ws to determine wheter in the vitro fertilization and culture of porcine oocyte using a hydrogel chamber were possible or not. Hydrogel chambers were made of polymerized 2-hydroxyethyl methacrylate. Matured follicular oocytes in Waymouth's medium and T L Hepes medium, tubal oocytes, and preincubated sperm in M199 medium were treansferred into the lumen of the hydrogel chambers. The chambers containing porcine oocytes and spermatozoa implanted into the mouse peritioneal cavity, and ova were examined after the recovery of the chambers at 84 hours after preservation start. The result was shown that fertilization and culture of porcine oocytes were successfully achieved inside of the hydrogel chamber.
Haque, Aminul;Bhuiyan, Mohammad Musharraf Uddin;Khatun, Momena;Shamsuddin, Mohammed
Journal of Embryo Transfer
/
v.26
no.2
/
pp.123-128
/
2011
In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.
This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.
This study was conducted to find out the effect of follicle size and oocyte type on in vitro maturation of poricine follicular oocytes. TCM-HEPEAS medium was used to basic medium, and the oocyte matured in vitro was stained with the Rapid staining method. The results obtained were summarized as follows; 1. The number of follicles an ovary was 20.5. The number of A-and B-typed oocytes an ovary was 2.34. The proportion of A-and b-types oocytes was 40% of the recovery oocytes. 2. Cumulus expanison indexes(CEI) by the follicle size were 1.62∼2.34(<2mm), 1.27∼2.28(2∼5mm) and 1.46∼2.75(>5mm). It was no differ to maturation rate by the follicle size. 3. The degree of oocyte maturation based on oocyte type did not differ for B-and C-typed oocyted but the index of oocyte type A was higher than that of b-and C-typed oocytes. 4. When follicluar oocytes were cultured for 42 hours, the proportion of the Met-II(second metaphase) stage were 22.5% (degree 1), 35.4%(degree 2) and 65.5% (degree 3).
Park, H.-S.;Jung, S.-Y.;Kim, T.-S.;Lee, M.-Y.;Jin, J.-I.;Hong, S.-P.;Lee, J.-S.;Kim, C.-H.
Journal of Embryo Transfer
/
v.19
no.2
/
pp.113-119
/
2004
The purpose of the present study was to examine whether collection time affects results of oocyte recovery from superovulated goats. Fiftyty-one mature Korean native goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen impregnated CIDR for 10 days and then the goats were divided into two groups. One group of the goats received a single intramuscular injection of 1,000 IU PMSG on Day 8 of CIDR insertion. The other group of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF_{2\alpha}$ on Day 8 and 400 IU hCG in the afternoon on Day 10. For oocyte recovery, donor goats were fasted 24 h before operation. Anesthesia was induced by intravenous injection of 2% xylazine(0.2 mg/kg body weight) and ketamin(11 mg/kg body weight). In vivo oocytes were recovered by follicle aspiration or oviduct flushing at 29 to 34, 35 to 40 and 41 to 50 h after hCG injection through mid-ventral incision. There was no significant difference in the mean number of CL and oocytes recovered. Oocyte collection at 29 to 40 h after hCG increased(P<0.05) the recovery rate of ovulated oocytes in oviducts compared to 41 to 50 h. The same results were also observed in the recovery of follicular oocytes. Oocyte grade was not affected by collection time. When oocytes were collected from follicular oocytes at 41 to 50 h after hCG, the recovery rate of Grade II oocytes was the lowest(P<0.05). From these results, it is suggested that oocyte recovery at 35 to 40 h after hCG will be successful for further use.
This study was carried out to determine the effect of cumulus cell attachment and various factors on in vitro maturation of pig foflicular oocytes. Oocytes with various configuration of cumulus cell mass were collected ftom ovaries of mature gilts by asperating with syringe equipped with needles of different gauges, follicle size and with or without cumulus cells. They were cultured in TCM-199 mediun containing FGS(fetal calf serum) for 30~48 hours in incubator with air containing 5% $CO_2$ at 38.5$^{\circ}C$. Mter orcein staining at in vitro maturation condition, GV, GVBD, anaphase, telophase and M II were observed. Results are surumarized as follows: 1. Recovery rates were 55.8, 55.5 and 34.4% when the cumulus-compacted oocytes were collected with 18, 21, 26 gauge needles of syringes, respectively. 2. 79% of oocytes with compacted cumulus cells were at GV stage and most of the oocytes with partially denuded and denuded cumulus cells were from GVBD to M- II stages. 3. Percentage of mature oocytes among those which are follicular diameter of 1~2, 3~6 and over 6 mm was 42.6, 53.2 and 60.8%, respectively. 4. Percentage of mature oocytes among those which are compacted, partially denuded and denuded was 60.5, 46.2 and 35.4% respectively. 5. Percentage of mature oocytes in co-cultured with monolayers of cumulus cells was higher (57.1%) than that found with oocytes cultured alone (53.4%).
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.