Phosphate가 제거된 단순배양액 중 아미노산의 첨가가 체외수정시술 후 임신율에 미치는 영향

이지삼,홍정의,유승환,정구성,홍기언,전은숙,허영문,이종인,Lee, Ji-Sam,Hong, Jeong-Eui,Yoo, Seung-Hwan,Jung, Goo-Sung,Hong, Ki-Eon,Jeon, Eun-Suk,Hur, Young-Mun,Lee, Jong-In 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2

The role of amino acids in culture media for IVF-ET was examined in a total of 76 cycles. Patients received clomiphene citrate (CC) followed by hMG or GnRH-a combined with gonadotropins (FSH/hMG) for controlled ovarian hyperstimulation. Severe male (<$4{\times}10^6$ motile sperm) or age factor (>39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. No significant differences were found in age, duration of infertility, follicle size, the level of $E_2$ on the day of hCG injection, the mean number of oocytes retrieved, total motile sperm count, fertilization rate and the mean number of embryos transferred between bHTF (without amino acids) and mHTF (with amino acids) groups. However, total ampules of gonadotropins were higher (p<0.01) in mHTF group than bHTF group. Significantly (p<0.05) more clinical pregnancies were recorded in mHTF group (13/30) compared with bHTF group (9/46). The multiple pregnancy rates were 11.1% in bHTF group and 7.7% in mHTF group. There were one ectopic pregnancy in mHTF group and one heterotopic pregnancy in bHTF group. Abortion rates were 22.2% in bHTF group and 7.7% in mHTF, respectively. The ongoing pregnancy or livebirth rate was significantly (p<0.05) higher in mHTF group (12/30) than bHTF group (7/46). These results suggest that the addition of amino acids in culture media is essential for culture of zygotes in vitro and adjustment of energy substrates in phosphate-free culture media appears to be beneficial for human IVF-ET procedure.

번식 1 ; 면양자궁의 PGF2α 분비에 대한 diacylglycerol 과 protein kinase C 의 역할

이지삼,William,J . Silvia 한국동물자원과학회 ( 구 한국축산학회 ) 1993 한국축산학회 심포지움자료집 Vol.- No.-

산양의 과배란 처리에 따른 발정발현과 단발정주기의 발생 및 발정기의 혈중 estradiol-17$\beta$의 농도변화

이지삼,박충생,최경문 한국동물번식학회 1990 Reproductive & developmental biology Vol.14 No.3

Behavioural estrus and short estrous cycles were observed and serum concentrations of estradiol-17$\beta$(E2) before and after of estrous were measured following superovulation treatments in 30 pluriparous Korean native goats. The goats were divided into 2 groups. Fifteen goats were injected IM with 1,000IU PMSG on Day 12 of the estrous cycle followed by 10mg PGF2$\alpha$ 48h later(P4+PMSG), and the other 15 goats were injected IM with 10mg progesterone(P4)in oil once daily for 10d beginning at any days of estrous cycle followed by 1,000IU PMSG and 10mg PGF2$\alpha$ at the 8th day of progesterone treatment(P4+PMSG group). After injection of PGF2$\alpha$, onset of standing estrus occurred in 12 of 15 goats(80.0%) at 50.0$\pm$7.7h and in 11 of 15 goats(73.3%) at 135.6$\pm$10.1h in PMSG and group and P4+PMSG group, respectively. The mean interval from PGF2$\alpha$ injection to first estrus was significantly(P<0.01) earlier in PMSG group than in P4+PMSG group. This result indicate that the delayed infusion of P4 in P4+PMSG group caused the later exhibition of their estrous behaviors. However, duration fo frist estrus(31.5$\pm$2.6h vs 26.2$\pm$2.3h), length of estrous cycle(14.1$\pm$3.3d vs 16.6$\pm$3.8d) and percentage of short estrous cycle(50.0% vs 45.5%) were not different between PMSG and P4+PMSG group. The mean concentration of serum E2 in 4 goats showing normal estrous cycle in P4+PMSG group(PP-NEC) was higher than in 6 goats showing normal(P-NEC) or in 6 goats showing short estrous cycle(P-SEC) in PMSG group. The peak level of serum E2 was observed at the time of onset of standing estrus in PP-NEC(67.6pg/ml), 6h earlier in P-NEC(53.1pg/ml) and 6h later in P-SEC(52.3pg/ml) than the onset of standing estrus. The profiles of serum concentration of E2 during the period of peri-estrus was similar in the goats of PMSG or P4+PMSG and also in the goats showing the subsequent estrous cycle of normal or short length.

山羊의 自然 및 誘起發情時 發情週期 및 發情持續時間에 관한 硏究

이지삼,송우준,최경문,박충생,Lee, Ji-Sam,Song, U-Jun,Choe, Gyeong-Mun,Park, Chung-Saeng 한국동물번식학회 1991 Reproductive & developmental biology Vol.15 No.1

This study was carried out to investigate the length of the estrous cycle and duration of estrus in Korean native goats. The effects of the type of last estrus : induced or natural, and the length of preceeding estrous cycle on the subsequent cycle and duration of estrus in goats were also examined. From 481 observations, the mean length of estrous cycles was 18.1${\pm}$0.5days. The cycle length was significantly(P<0.05) shorter following induced estrus (15.2${\pm}$0.8 d) than natural estrus(19.0${\pm}$0.6 d). Significantly higher(P<0.05) incidence of short estrous cycle was observed following induced estrus(40%) than natural estrus(27%). The frequency distribution in the estrous cycle was 30%, 3%, 56% and 11% for short(3-11 d), medium(12-16 d), normal(17-24 d) and long(${\geqq}$25 d), respectively. The most frequent type of short estrous cycle was 6 days in length. Mean duration of estrus was 34.0${\pm}$0.5 h with a range of 18 to 84 h. Duration of estrus was not significantly different the preceeding natural (33.8${\pm}$0.6 h) and induced estrus (34.4${\pm}$1.1 h), and was not significantly affected by the last cycle length. These results suggest that the short estrus cycles are more frequent following induced estrus than natural one, and the duration of estrus are affected greatly by the various intrinsic and extrinsic factors.

재래산양의 계절적 무발정기의 혈중 Progesterone 의 변화

이지삼,곽대오,박충생 ( J . S . Lee,D . O . Kwack,C . S . Park ) 한국축산학회 1985 한국축산학회지 Vol.27 No.12

This experiment was conducted to investigate the breeding seasonality in Korean native goats. Nineteen primiparous goats of 2-years old and about 23㎏ of body weight were barn-housed at Gyeongsang National University Farm(N.L.: 35°1`) and allowed with daily supplements of about 200g and liberal feeding of grasses through the experimental period from November, 1982 to October, 1983. The goats were teased twice daily for 30 minutes in the morning(06:00) and evening(18:00) with two vasectomized bucks and behavioural estrus was recorded. For the effect of prostaglandin F₂α(PGF₂α) treatment on the induction of estrus, 10 anestrous goats were given intramuscular injection of 3㎎ PGF₂α(Lutalyse, Upjohn Co.) at a certain time of anestrus. Jugular vein blood was collected from venepuncture every 3 days from July 8 to August 7 for determination of serum progesterone concentration by radioimmunoassay. Eight of 10 anestrous goats were injected with PGF₂α in August. Two of 8 treated goats, Goat 29 and Goat 41 exhibited standing estrus in 12 and 24 hours after PGF₂α treatment. Serum progesterone concentration during anestrous period was ranged from 0.05 to 0.44 ng/㎖ except for a short period in 3 goats. Such levels of serum progesterone concentration are assumed much lower level compared to those during the luteal phase of estrous cycle of goats in breeding season. These data suggest that Korean native goats have a nonbreeding season of which duration may be varied among the individuals. The incidence of anestrous goats was found to be may frequent in summer than in any other season.

Effects of Chronic Administration of an Oxytocin Antagonist on Luteal Function and Uterine Secretion of Prostaglandin F_2α in the Ewe

Lee, J. S.,Park, H. S.,Chung, J. Y.,Silvia, William J. 진주산업대학교 농업기술연구소 1997 農業技術硏究所報 Vol.10 No.-

본 실험은 면양의 황체기능 및 자궁에서의 PGF_2α의 분비에 미치는 oxytocin antagonist의 효과를 알아보기 위하여 번식기에 있는 24두의 면양을 공시하여 발정주기의 0∼3일, 또는 11∼16일에 oxytocin antagonist를 경정맥으로 투여한 후 발정주기의 기간과 혈중 PGF_2α의 농도변화를 조사하였고, 발정주기의 제15일에 oxytocin 10 IU를 경정맥으로 투여한 후의 혈중 PGF_2α의 농도변화를 조사하였다. 발정주기의 초기나 말기에 oxytocin antagonist의 투여는 황체기능에 유의적인 영향을 미치지 못하였고, 혈중 PGF_2α의 수준에서도 현저한 반응이 관찰되지 않았다. 그러나 발정주기의 말기에 oxytocin의 투여는 자궁에서의 PGF_2α분비를 촉진시켰다. 이러한 결과는 oxytocin의 투여에 의한 PGF_2α의 분비증가는 발정주기의 말기에 나타난다는 사실을 입증한다. This study was conducted to investigate the effects of oxytocin antagonist(OTA) on ovine luteal function and release of uterine prostaglandin F_2α(PGF_2α). Twenty-four crossbred ewes were randomly assigned to 1 of 4 treatment groups. Oxytocin antagonist(0.1㎍/day/ewe) or saline was administered via intravenous route on Day 0∼3 or Day 11∼16 of the estrous cycle. Concentrations of PGFM in serum samples were analyzed by RIA. The length of estrous cycle was not affected by OTA treatment. Increased concentrations of PGFM in serum samples after oxytocin(OT) challenge were observed from 5 min after OT injection. Peak levels were found at 20 min after OT injection.

Changes in Serum Concentrations of Progesterone during Estrous Cycle after Chronic Administration of An Oxytocin Antagonist in the Ewe

Lee, Ji Sam,Silvia, William J.,Park, Hee Sung,Chung, Jang Yong 진주산업대학교 농업기술연구소 1998 農業技術硏究所報 Vol.11 No.-

본 실험은 번식기에 있는 24두의 면양을 공시하여 발정주기의 0-3일, 또는 11-16일에 oxytocin antagonist를 경정맥으로 투여한 후 황체기능 및 혈중 progesterone의 농도변화를 조사하기 위하여 실시하였다. 발정주기의 초기나 말기에 oxytocin antagonist의 투여는 황체기능에 유의적인 영향을 미치지 못하였고, 혈중 progesterone의 농도에서도 현저한 반응이 나타나지 않았다. 이러한 결과는 면양의 초기 또는 말기의 황체는 oxytocin antagonist에 대하여 능동적인 반응을 나타내지 못하며, 이러한 황체기능의 조절에는 다른 hormones에 의한 복합적인 내분비 기전이 작용할 수 있음을 나타낸다. This experiment was performed to investigate the effects of an oxytocin antagonist(OTA) on luteal function and change in serum concentrations of progesterone in the ewe. Twenty-four crossbred ewes were randomly assigned to 1 of 4 treatment groups. An antagonist of oxytocin(0.1g/day/ewe) or saline was administered via intravenous route on day 0-3 or Day 11-16 of the estrous cycle. Serum concentrations of progesterone and 13, 14-dihydro-15-keto-PGF_2x(PGFM) were analyzed by RIA. The mean length of estrous cycle was not affected by OTA treatment. Concentrations of progesterone and PGFM were not different between OTA-treated and control ewes. In conclusion, our results imply that early or late corpus luteum(CL) may be inactive in response to OTA and other factors can be involved in early development or late maintenance of CL function.

자궁강내 인공수정에 의한 임신율

홍정의,이지삼,Hong, Jeong-Eui,Lee, Ji-Sam 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.2

The effectiveness of intrauterine insemination (IUI) combined with controlled ovanan hyperstimulation (COH) in the treatment of infertility with various etiologies was compared in a total of 152 cycles. Patients received a maximum of three IUI cycles for the treatment. Severe male ($<2\times10^6$ motile sperm) or age factor (> 39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac was seen on ultrasound. The overall clinical pregnancy rate was 7.9% per cycle (12/152) and 9.7% per patient (12/124). The pregnancy rates were 0% in unstimulated natural (0/18), 7.5% in CC (3/40), 8.2% in CC+hMG (4/49), 5.9% in GnRH-a ultrashort (1/17), 5.9% in GnRH-a long (1/17) and 27.3% in dual suppression cycles (3/11), respectively. The pregnancy rate was higher in dual suppression cycle than other stimulated cycles, but this was not significant. The multiple pregnancy rates were 25.0% (2 twins and 1 triplet). No patient developed ovarian hyperstimulation. Abortion rates were 66.7% in CC (2/3) and 100% in ultrashort cycles (1/1). The livebirth rate was 5.9% per cycle (9/152) and 7.3% per patient (9/124). There were no differences in age, duration of infertility, follicle size, total ampules of gonadotropins and days of stimulation between pregnant and non-pregnant groups. However, significant(P<0.05) differences were observed in the level of estradiol $(E_2)$ on the day of hCG injection ($3,266.6{\pm}214.2$ vs $2,202.7{\pm}139.4$ pg/ml) and total motile sperm count ($212.1{\pm}63.4$ vs $105.1{\pm}9.9\times10^6$) between pregnant group and non-pregnant group. These results suggest that IUI combined with successful ovarian stimulation tends to improve the chance of pregnancy as compared to IUI without COH and a total motile sperm count may be considered predictive of the success for pregnancy.

유전자 분석을 통하여 선발된 한우로부터 초음파 유래 체외수정란 이식에 의한 고품질 한우 생산기술의 실용화 2 : DNA 검정우로부터 초음파 유래 체외수정란의 생산에 관한 연구

박희성,이지삼,진동인,박준규,홍승표,이명열,정장용 韓國受精卵移植學會 2001 한국동물생명공학회지 Vol.16 No.3

본 연구에서는 DNA marker가 검정된 한우로부터 생산한 체외수정란을 이식하여 육질 및 육량의 유전적 능력이 우수한 한우를 대량생산하여 고품질 한우 쇠고기 생산 시스템을 구축하기 위한 전단계로서 DNA marker 검정 한우로부터 초음파유도 난포란을 채란하여 체외수성 및 수정란의 체외 발달에 미치는 각종 요인들과 배반포기 수정란의 부화율 개선을 위하여 투명대를 laser로 drilling을 실시하여 부화율을 조사하였다. 초음파유래 체외수정란의 분할률 This study was designed to examine the factors affecting in fertilization and development of embryos in vitro, and to examine whether zone drilling by laser irradiation can improve the hatching rate of IVF embryos from DNA marker-proved Hanwoo. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local anesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analyzed using the GLM procedure of SAS. Sperm separation methods did not have any significant effect on cleavage or developmental abilities of IVF embryos. Significantly(P<0.05) higher cleavage rate was observed in embryos from GI(60.0%, 3/5), GII(69.2%, 18/26) and GIII(62.1%, 59/95) compared to embryos from GIV oocytes(36.2%, 25/69). And the developmental rate to blastocyst stage was higher(P<0.05) in embryos from GI(33.3%, 1/3) and GII oocytes(38.9%, 7/18) than those from GIII(16.9%,10/59) and GIV oocytes(4.0%, 1/25). There was no significant difference in development of IVF embryos to blastocyst by media for in vitro culture. Proportion of hatched blastocyst was significantly(P<0.05) higher in embryos received zona drilling by laser than those of non-drilled.

유전자 분석을 통하여 선발된 한우로부터 초음파 유래 체외수정란 이식에 의한 고품질 한우 생산기술의 실용화 1 : DNA 검정우에서 초음파기기를 이용한 난포란의 채란에 관한 연구

박희성,이지삼,진종인,박준규,홍승표,이명열,정장용 韓國受精卵移植學會 2001 한국동물생명공학회지 Vol.16 No.3

본 연구는 고품질육의 DNA marker가 규떵된 한우로부터 초음파유노 난포란의 연속적 채취를 통하여 능력이 우수한 한우 수정란온 대량생산하는 방법의 확립과 이를 한우농가에 응용하고자 초음파 난자채취기를 이용하여 등지방층두께, 일당증체량, 근내지방도 및배최장근 단면적에 연관된 DNA marker를 보유하고 있는 한우 5두로부터 개체및 난포수, 채취방법, 회수한 난포란의 등급 등을 조사하였다. 한우 5두의 개체별 난포수는 6, 10, 5, 4 및 11회 This study was designed to establish the superior method for IVF embryos from DNA marker-proved Hanwoo cattle. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from unstimulated. immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local abesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analysed using the GLM procedure of SAS. Mean number of follicles identified on ultrasound was 5.5 2.9 in right and 4.3 2.8 in left ovaries, respectively. The highest follicles(16.62.6) were found in 5101 cow compared to others. Recovery rate of follicular oocytes in individual cow was highest in 5101 cow with 89.3% in > 2mm and 94.0% in 2mm follicles. Total recovery rate was significantly(P<0.01) higher in 2mm(85.7%, 130/154) than > 2mm follicles(74.2%, 201/271). Significantly more oocytcs of Grade IV were recovered from > 2mm follicles. Mean number follicles recovered was 4.83.7. 3.03.4 and 0.30.6 in 2mm, 2~6mm and 6mm follicles. respectively. Our results imply that the more fertilizablc oocytes can be recovered from invisible-immature follicles by the combination of simultaneous rectal ovarian-palpation and ultrasound-guided approach in Hanwoo cattle.