젖소의 초음파 유도 채란율에 대한 FSH 전처리 효과의 비교

박충생,조성근,이정규,강태영,박성재,공일근,최민철,Park, Chung-Saeng,Jo, Seong-Geun,Lee, Jeong-Gyu,Gang, Tae-Yeong,Park, Seong-Jae,Gong, Il-Geun,Choe, Min-Cheol 한국동물번식학회 1997 Reproductive & developmental biology Vol.21 No.2

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.

Studies on nuclear transplantation in mouse embryos II. Developmental potential of nuclei from embryos of different developmental stages

박충생,최상용,이효종,박희성,Park, Choong-saeng,Choe, Sang-yong,Lee, Hyo-jong,Park, Hee-sung The Korean Society of Veterinary Science 1990 大韓獸醫學會誌 Vol.30 No.4

Single nuclei from two-, four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos by micromanipulation and Sendai virus mediated fusion. The developmental potential of these reconstituted embryos in vitro and in vivo was examined. It was found that the single nuclei which were transplanted to enucleated two-cell embryos were not only able to develop to the blastocyst stage in vitro(two-cell nuclei, 76.5%; four-cell nuclei, 68.4%; eight-cell nuclei, 48.3%), but also able to develop to full term in vivo after transfer to recipient mice(two-cell nuclei, 37.1%; four-cell nuclei, 29.6%; eight-cell nuclei, 16.3%). Although the proportion of live young produced after transfer of nucler of nuclear transplant embryos which received eight-cell nuclei was significantly (p<0.05) reduced, it would be suggested that the overall efficiency in producing identical offspring is greater when eight-cell embryos were selected for nuclear donor than two- or four-cell embryos were selected. 포유동물의 초기 발생단계에서 핵의 분화와 전능성(totipotency) 을 규명하고, 수정란의 cloning technique를 개발하여 우량유전자로 조성된 개체를 복제함으로써 효과적인 종축개량 기법으로 응용하기 위하여 생쥐 수정란을 모델로 하여 미세조작기법과 Sendai virus를 이용한 핵융합기술을 이용하여 인위적으로 동일한 유전자를 가진 복제 수정란을 작출하고 이들의 작출효과, 체외발달능력 및 체내 이식후 개체발생여부 등을 조사하였다. 2-세포기, 4-세포기 및 8-세포기의 수정란으로부터 핵을 채취하여 이들을 탈핵된 2-세포기의 수정란에 이식하였을 때, 이들의 핵융합 성공율은 각각 88.6%, 87.1% 및 84.7%이었다. 나아가서 이들 핵융합된 수정란을 체외에서 96시간 배양한 결과, 2-세포기, 4-세포기 및 8-세포기의 핵이 이식된 수정란은 각각 76.5%, 68.4% 및 48.3%가 배반포로 발달하였다. 핵이식 후 체외에서 배반포로 발달된 수정란을 골라 수란생쥐에 이식하였던 바, 2-세포기의 핵이 이식된 수정란 156개 중 58개(37.1%) 가 발달하여 신생자로 생산되었으며, 4-세포기의 핵아 이식된 수정란 135개 중 40개(29.6%)가, 그리고 8-세포기의 핵이 이식된 92개의 수정란 중 15개(16.3%)가 신생자로 생산되었다.

산양의 수정란 이식 및 조작기법 개발에 관한 연구 Ⅰ. 산양의 발정유기 및 동기화에 관하여

박충생(C . S . Park),최상용(S . Y . Choe),이효종(H . J . Lee),이지삼(J . S . Lee) 한국축산학회 1989 한국축산학회지 Vol.31 No.1

This experiment was conducted to find out an efficient method for estrus induction and synchronization in goats. Sixty of 83 pluriparous Korean native goats received a single i.m. injection of 3㎎ PGF2α on Days 3 to 12 of the estrous cycle. Remained 23 goats received twice daily i.m. injection of 5㎎ progesterone for 6 or 7 days and a single i. m. injection of 3㎎ PGF2α 1 day before the last injection of progesterone. Serum progesterone concentrations were analyzed by RIA. A single intramuscular injection of 3㎎ prostaglandin F2α(PGE2α) on Days 5 to 12 of the estrous cycle induced estrus in 96% of 50 goats treated, but this treatment on Day 3 to 4 was not effective. Estrus was induced in all of 23 goats injected 3㎎ PGF 2αi.m. 24 hours before the last one of successive injections of 5mg progesterone for 6 or 7 days. The onset of estrus was significantly(P$lt;0.01) earlier in PGF 2α treated goats during Days 5 to 6 of the estrous cycle than those treated on Days 7 to 12. PGF 2α treatment with progesterone delayed significantly (P$lt;0.01) the onset of returning to estrus, compared with the PGF2α injection only during mid-luteal phase of the cycle. Serum progesterone concentration was decreased to a nadir at 24 hours following PGF2α administration on Days 5 to 8 of the cycle, but increased to 2.39±0.66 to 2.69±0.56ng / ml at 48 hours after PGF2α injection on Days 3 to 4. Incidence of short estrous cycle following estrus synchronization by PGF2α with or without progesterone was 26.1 % and 26.0 %, respectively. More effective synchronization of estrus was feasible by progesterone treatment for 6 or 7 days plus PGF2α injection 24 hours prior to the last progesterone administration, compared with a single intramuscular injection of 3㎎ PGF2α alone.

산양의 수정란 이식 및 조작기법 개발에 관한 연구 2 . 산양의 분만 및 유산 후의 단발정주기

박충생(C . S . Park),최상용(S . Y . Choe),이효종(H . J . Lee),이지삼(J . S . Lee) 한국축산학회 1991 한국축산학회지 Vol.33 No.4

This experiment was conducted to investigate the incidence of short estrous cycle and control of short cycle in Korean native goats. Abortion was induced by a single i.m. injection of 3 ㎎ PGF₂α at late stage of gestation. Does after parturition or abortion were teased twice daily with vasectornized bucks and behavioral estrus was recorded. Serum progesterone concentration was analysed by RIA. Mean post-partum or post-abortion interval to first estrus was shortest as 11.9 days in PGF₂α-aborted does compared to 22.1 days in does isolated from their kids within 7 days after parturition and 26.8 days in suckling does. Duration of this first standing estrus was not significantly different among groups. Four limes daily injections of 25 ㎍ Gn RH to does for Day 3 to 5 of the first estrous cycle following parturition or abortion ruduced significantly(p$lt;0.0l) the incidence of short estrous cycle to 38.5% compared to 80.0% in control group of does. Mean concentration of serum progesterone in does showing short estrous cycle was found to be basal level at estrual phase but considerably high level up to 1.06 ㎎/㎖ on Day 3 to 4 of the cycle. These results indicate that the short estrous. cycle may he resulted from the premature luteal regression and GnRH treatment during early luteal phase of the estrous cycle can partly prolong the short lifespan of corpora lutea.

산양의 수정란 이식 및 조작기법 개발에 관한 연구 4 . 생쥐 및 산양 수정란의 분할조작에 의한 일란성 쌍태유기

박충생(C . S . Park),최상용(S . Y . Choe),이효종(H . J . Lee),이지삼(J . S . Lee),박희성(H . S . Park) 한국축산학회 1991 한국축산학회지 Vol.33 No.4

This experiment was conducted to establish a simple technique for bisecting mouse and goat embryos of morula to blastocyst stage and to develop a better culture and transfer system of demi-embryos for the production of identical twins. Mouse morulae or blastocysts and goat blastocysts were easily bisected without pronase treatment, using a microblade fixed to a micromanipulator. Bisected mouse embryos developing normally during culture were 71.4% of a total of 319 morulae and blastocysts. Survival rate of demi-embryos(29.0%) after culture and surgical transfer to recipient mice was comparable to result of intact embryos(26.0%). More transferable embryos (3.5±0.8/donor) were recovered in 7 to 8 days after mating when the does were induced to superovulate by injection of FSH-p in decreasing doses and progesterone priming for 10 days. and this superovulation regimen resulted in very low incidence of short estrous cycle(6.7%). Three kids were produced from 2 of 6 does received intact embryos. Fourteen pairs and one-half of demi-embryos were transferred to 15 recipients. of which one recipient delivered a single female kid.

산양의 수정란 이식 및 조작기법 개발에 관한 연구 5 . 생쥐 및 산양 분할 수정란의 생존성 및 이식 후 수태율의 향상

박충생(C . S . Park),최상용(S . Y . Choe),이효종(H . J . Lee),이지삼(J . S . Lee),박희성(H . S . Park) 한국축산학회 1991 한국축산학회지 Vol.33 No.5

This experiment was conducted to compare different procedures for improvement of viability of embryos after bisection and to investigate some factors affecting development of the bisected embryos. Demi-embryos were successfully produced by micromanipulation of mouse and goat embryos at stages from morula to blastocyst. In mouse, a superior viability(90%) of demi-embryos was achieved by culturing expanded blastocysts after bisection. The highest pregnancy rate(57.1%) and implantation rate(28.2%), which were almost same as the rates of intact embryos. were also achieved by transferring the demi-embryos at this stage after culture for 24 h in vitro. Twenty -eight 3%) of 30 embryos collected from superovulated Korean native goats were successfully bisected, of them 23 demi-pairs were transferred to recipients. Six and 3 does of them were diagnosed pregnant on 21 and 60 days of gestation, respectively. The present result indicates that in goat, hatched blastocysts might be more suitable for successful bisection to produce monozygotic twins than morulae or earlier blastocysts.

산양의 수정란 이식 및 조작기법 개발에 관한 연구 3 . PMSG 와 FSH 를 이용한 산양의 과배란 유기

박충생(C . S . Park),최상용(S . Y . Choe),이효종(H . J . Lee),이지삼(J . S . Lee) 한국축산학회 1991 한국축산학회지 Vol.33 No.4

This study was conducted to investigate the effects of PMSG or FSH treatment with PGF₂α to induce superovulation in Korean native goats. Does were treated with a single s.c. injection of 1,000 IU PMSG on Day 12 or twice daily s.c. injections of a total of 20 ㎎ FSH in decreasing doses over 4 days beginning on Day 12 of the estrous cycle, Estrus was induced by a single i.m. injection of 10 ㎎ PGF₂α 48 hours after the first gonadotropins injection. The does in estrus were mated with intact bucks twice daily 12 hours apart. Ovarian responses and embryo development were observed by laparotomv 72 hours after mating. Mean interval from PGF₂α injection to onset of estrus was significantly(p$lt;0.01) shorter (45.2±7.5 hours) in FSH group than ill PMSG group(64.8±9.6 hours). Duration of estrus, length of estrous cycle and the incidence of short estrous cycle following superovulation were not significantly different between the 2 treatment groups. The mean number of corpora lutea(P$lt;0.01) and ma recovered(P$lt;0.05) were significantly greater for FSH group(13.2 and 9.2) than for PMSG group(5.1 and 3.5). The mean number of trans-ferable embryos kva, 5.9 and 1.9 in FSH-treated and PMSG-treated group. respectively. These results indicate that FSH treatment with PGF₂α could be superior to PMSG treatment with PGF₂α for the induction of superovulation in Korean native goats.

Silver Nitrate 용액의 정소내 주입에 의한 가토의 거세효과에 관한 연구

이은봉,박충생,강대진,윤창현 ( E . B . Lee,C . S . Park,D . J . Kang,C . H . Yun ) 한국축산학회 1985 한국축산학회지 Vol.27 No.12

This experiment was carried out to develop a chemical castration with a single intratesticular injection of silver nitrate solution in domestic rabbits. A total of 44 bucks of 4 to 5 months old were alloted at random into the following 4 groups: 9 bucks for controls, 6 bucks for 0.3㎖ injection of 1% AgNO₃, 14 bucks for 0.5㎖ injection of 1% AgNO₃ and 15 bucks for 0.5㎖ injection of 5% AgNO₃. Changes in body weight, testicular width in scrotum. weight and histology of testis at necropsy, blood cell counts and plasma levels of testosterone of the buck were measured for 4 weeks after treatment. The results obtained were summarized as follows: Compared with the controls, the body weight gain of bucks treated with AgNO₃ was not significantly (P$lt;0.05) different during the experimental period for 4 weeks. The white blood cell counts were tended to be increased slightly but insignificantly 1 day after injection of AgNO₃ and then decreased to normal level soon. There was no significant change in red blood cell counts in the bucks treated with AgNO₃. Most of the testis of bucks were swollen significantly (P$lt;0.05) by 6 to 12㎜ in the width of testis in scrotum 1 day after injection of AgNO₃, but began to decrease in sue from 3 days after treatment and to their normal size in a week. The AgNO₃ injection was not sufficient to castrate some bucks successfully. Only 60.0 to 66.7% of the bucks treated with 0.0㎖ of 1 or 5% AgNO₃ solution showed the plasma levels of testosterone of !sower than 0.1 ng/㎖ by 28 days after treatment. This result was considered to be due to the fact that not a whole but a limited part of testis was injured by the present method of injection. The weight of testis of the bucks of which plasma testosterone were less than 0.1 ng/㎖ by 7 to 28 days after treatment was not significantly(P$lt;0.05) different between the treatments or periods after treatment. This result indicates that the period of 4 weeks following treatment might be insufficient for the injured testicular tissue to be necrotized and absorbed fully. The extent of damage of testis by injection of AgNO₃ was much different by the local effect in the parts of distance from injection. The proximal portion of testis injected with AgNO₃ was fully damaged and necrotized, but a distal part of testis was not severely damaged.

Studies on nuclear transplantation in mouse embryos. I. Functional differences between maternal and paternal genomes

최상용,박충생,이효종,박희성,Choe, Sang-yong,Park, Choon-saeng,Lee, Hyo-jong,Park, Hee-sung The Korean Society of Veterinary Science 1990 大韓獸醫學會誌 Vol.30 No.2

모성 및 부성 genome의 기능을 알아보기 위하여 미세조작기법과 Sendai virus를 이용한 핵융합 기술을 이용하여 2개의 자성전핵만으로 구성된 2배체의 gynogenetic 수정란을 그리고 2개의 웅성전핵만으로 구성된 2배체의 androgenetic 수정란을 인위적으로 작출하였다. 이들의 작출효율은 biparental 수정란에서는 56%, gynogenetic 수정란에서는 50% 그리고 androgenetic 수정란에서는 56% 이었다. 이들을 체외에서 배양한 결과 gynogenetic 및 androgenetic 수정란은 2-세포기 이후에는 biparental 및 intact 수정란에 비하여 그 발달능이 매우 저조하였으나 이들 중 25% 이상이 포배까지 발달한 수 있음을 확인하였다. Gynogenetic 및 androgenetic 수정란을 동기화된 수란생쥐의 난관내에 이식하였던 바, androgenetic 수정란은 전혀 착상 되지 않았으나, gynogenetic 수정란에서는 착상이 확인되었다. 핵이식기법으로 인위조작된 2배체의 biparental 수정란으로부터 28마리의 생쥐 신생자를 생산하였다. By nuclear transplantation technology twenty eight mice have been produced after transfer of heterozygous biparental eggs. Also heterozygous gynogenetic eggs with two female pronuclei and heterozygous androgenetic eggs with two male pronuclei have been obtained by injecting a male or female pronucleus with Sendai virus into the perivitelline space of enucleated haploid zygotes at pronuclear stage. The success rate of enucleation, karyoplast injection and fusion of both the pronuclei was 80.3, 83.4 and 81.8%, respectively. The overall pronuclei fusion rates by this technique were 56, 50 and 56% in biparental, gynogenetic and androgenetic eggs, respectively. The evidence was ascertained that the gynogenetic and androgenetic eggs were also able to develop in vitro up to blastocyst stage, even though their developmental potential was greatly diminished beyond 2-cell stage. The gynogenetic eggs were able to develop in vivo up to day 10 of pregnancy, while the androgenetic eggs failed to develop in vivo during the same period.

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.