The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes () was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage () and mean number of cells in blastocyst ( cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 FSH,i numectivelo. In SCNT, fusion () of cell-cytoplast couplets and siosequent embryo cleavage () were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 FSHr(25% vs. ). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

본 연구는 체외 성숙된 난자와 동결 융해 정자를 이용한 돼지의 체외 수정 과정에서 난구 세포의 존재가 정자 침투율, 웅성전핵 형성률 그리고 후기배로의 체외 발육에 미치는 영향을 알아보기 위하여 수행되었다. 돼지 난소로부터 난자-난구세포 복합체를 채취하여 eCG/hCG, 10% 돼지 난포액, epidermal growth factor 등이 첨가된 TCM 199 배양액에서 44시간 배양하여 체외 성숙을 유도하였다. 성숙 배양 후 난구 세포를 제거한 난자와

The aims of these study were to diagnose early pregnancy and reproductive disorders by using progesterone concentration and ultrasonography. The measurement of blood progesterone (P) concentration was conducted to diagnose pregnancy and to detect corpus luteum (CL) or evaluate disorder of CLs. As a result, the incidence rates of reproductive disorders were as follows : SH and EED (41.9%), inacitve ovaries (32.6%), follicullar cyst (9.3%), PCL (7.0%), endometritis (4.7%), pyometra (2.3%) and luteal cyst (2.3%). 61 Cows having Pconcentration 1.0 ng/ml(at the insemination) were increased to 1.0 ng/ml 6day after insemination. 50 cows among 61 cows were diagnosed pregnant. 8 cows among 13 HanWoos having Pconcentration 1.0 ng/ml at the insemination and 1.0 ng/mnl 6 day after insemination had non-ovulatory estrus and the others had Pconcentration 1.0 ng/ml at the insemination and 1.0 ng/ml 6 day after insemination, which was the error of estrus detection. All 13 cows were diagnosed non-pregnant. 47 cows diagnosed pregnant after insemination of Pconcentration 3.0 ng/ml were examined by ultrasonography at 30 day post-insemination. As a result, 41 cows were diagnosed pregnant (87.2%) but 14 cows having Pconcentration 3.0 ng/ml at 21 day after insemination was diagnosed to non-pregnancy. Calving intervals by surveying 100 cows were as follows 11~12 months (20%), 12~13 months (36%), 13~14 months (19%), 14 months (25%), respectively. In conclusion, hormone and ultrasonography help to detect reproductive disorders exactly and diagnose early pregnancy. This study suggest that diagnosis of early pregnancy and reproductive disorder by blood Pconcentration and ultrasonography improve reproduction management of HanWoo.

The objective of this study was to compare different superovulation treatments using PMSG or PG600 and to determine the optimal time of oocyte recovery after hCG administration. A total of 90 prepubertal Yorkshire x Landrace gilts crossed with Duroc, 6~7 months old and 100~120 kg of body weight, were used. PMSG (1,500 IU/head) or 5~7.5 ml of PG600(400 IU of PMSG and 200 IU of hCG) were administrated subcutaneously, and then 1,000 IU of hCG were administered intramuscularly at 72 hours after PMSG or PG600 injection. At carious time of 44, 46, 48 and 50 hours after hCG injection, superovulated gilts were slaughtered in a local abattoir. Ovaries together with oviducts were excised from the body immediately after slaughtered and transported to laboratory in 39 saline. Ovaries were examined fur the number of corpus hemorrhagicum and unovulated follicles present in the surface of ovary. The unovulated follicles were categorized into small (1~3 mm in diameter) and large (4~8 mm) groups according to their diameter. Oocytes were recovered by flushing both oviducts with micropipette tip (1~100 l) attached to a 10-ml disposable syringe. The number of CH on ovary and recovered oocytes at 46, 48 and 50 hr after hCG injection in PG600 treated groups were significantly higher than the other group. Group of phCG 50 hr among PMSG treated groups had a greater number of CH and recovered oocytes(P<0.05). The number of CH on ovary and recovered oocytes at 50 hr after hCG injection in 1 vial(7.5 ml) of PG600 treated groups was significantly higher than 1 vial(5 ml) of PG600 treated group(P<0.05). In conclusions, considering a number of corpus hemorrhagicum and recovered oocytes after superovulation in gilts, effective time of oocyte recovery by treatment with PMSG and hCG was post-hCG 50 hr and with PG600 plus hCG was post-hCG 46, 48 and 50 hr. Also, admini-stration of 1 vial(7.5 ml) of PG600 treated group had a great number of CH and recovered oocytes.covered oocytes.

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 /mL), and higher rate of encleation was obtained at 7.5 and 15 /mL than at /mL. In Experiment 2, oocytes enucleated in 7.5 /mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 ) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 or 90 , but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

핵 이식에 공여되는 수핵난자의 효과적인 동결보존을 위하여 한우 성숙난자를 1.0 M dimethylsulfoxide(DMSO) 또는 1.0 M glycerol이 함유된 동결보호제를 이용하여 처리하거나, 동결보호제 처리 후 완만동결법을 이용한 동결융핼르 시행하여 상기 실험처리로 야기되는 투명대 경화현상을 관찰하였다. 도축장 유래의 난소에서 미성숙난자를 채취한 후 10% 소 태아혈청을 함유한 TCM-199을 이용하여 22∼24 시간 동안 체외성숙배양을 이해

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

Real time B-mode ultrasound was used to detect the early conceptus in 187 Korean native cattles between days 10 and 60 after last insemination. The ultrasound diagnostic findings were systemically confirmed by palpation per rectum after the 60th day of last insemination. The embryonic vesicle and the embryo proper within the veside were first visible on mean day fl and 23, respectively. The heartbeat of the embryo proper could be detected on day 26, and the limb buds, placentomes, amnion, fetal movement, umbilical cord, optic area and split hooves were first visible on day 33, 34, 34, 44.5, 45, 32 and 48, respectively. The mean length of embryo proper was 3.8mm on day 23 which later increased to 56. 6rnrn on day 60. When ultrasound was used to detect the conceptus between days 20 and 30 after insemination and palpation per rectum after the 60th day of insemination, the accuracy rates of pregnancy detection by ultrasound scanning at days 20, 22, 24, 26, 28, 30 were 44.4, 69.2, 78.6, 87.5, 90.0, 93.3%. In summary, the early pregnancy diagnosis of Korean native cattle with ultrasound appears high accuracy rates. It is considered that ultrasound can be used in veterinary practice well.

The present study was undertaken to examine the critical effect of + 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 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 sec in electroporation media(0.28 M mannito' and PBS) with different + 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 +(60.3, 82.2 and 75.0%) were significantly higher than without +(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 +(71.0, 75.8 and 75.4%) were significantly higher than without +(23.5%) after post-stimulation incubation(p<0.005). After incubation of oocytes in SOF with and without + following electric stimulation in 0.28 M mannitol with 0.10 mM +, the rates of pronuclear formation of bovine oocytes in +-free SOF(85.7%) was significantly higher than in SOF with 1.71 mM +(62.5%, p<0.05). When oocytes were stimulated in two electrostimulation media supplemented with + and incubated in +-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 +. Furthermore, to improve the activation rates, it was hetter that stimulated oocytes were incubated in +-free SOF after electric stimulation than in SOF with +.

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 g /mL of FSH and LH and 1 g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

본 실험은 일당귀의 개화 후 일수에 따른 종자 등숙 특성을 알아보고자 수행하였다. 2019년 농촌진흥청 약용작물과 시험 포장에서 채종한 종자를 시험재료로 사용하였다. 개화 후 일수에 따라 종자 무게와 발아율이 조사되었고, 등숙 과정 동안 종자 내에서 배종비(E:S ratio)가 측정되었다. 결과적으로는 각 소화서마다 개화 후 일수가 증가할수록 종자 무게가 유의적으로 증가하였으며, 각 소화서에서 발아가 시작되는 시기는 차이가 있었다. 또한 종자 내에서 배의 길이는 계속해서 성장하여 배종비가 높아지는 것을 관찰하였다. 일당귀는 다양한 소화서에서 꽃이 피기 때문에 종자의 배종비가 종자 발아에 영향을 미칠 수 있다. 본 연구를 바탕으로 일당귀의 우량 종자 생산을 위해서는 개화 후 50일부터 70일경이 가장 적합한 것으로 사료된다.

Background : In previous study, we reported Sclerotium rot caused by Sclerotium rolfsii in Ixeridium dentatum for the first time. This experiment was conducted to select highly effective pesticides against Sclerotium rot caused by S. rolfsii in I. dentatum. Methods and Results : The chemical efficacy and the injury test were carried out. A total of five pesticides were used for the experiment test. For the efficacy test, we investigated spore germination and mycelial growth inhibiting ability by each pesticides in vitro and disease inhibiting ability in the field. For the chemical injury, we investigated appearance of abnormalities on condition of reference amount and fold amount in the field. In vitro, three kinds of chemicals such as Fludioxonil suspension concentrate (SC), Tebuconazole suspension concentrate (SC), and Flutolanil emulsifiable concentrate (EC) showed complete spore germination inhibitory effect, However in two chemicals such as Pyraclostrobin water-dispersible granule (WG) and Pyribencarb suspension concentrate (SC), the mycelial growth inhibitory effect was partially recognized but the spore germination was not inhibited. In the field, we performed an artificial inoculation experiment using sclerotia. As a result four kinds of chemicals such as Fludioxonil SC, Tebuconazole SC, Flutolanil EC, and Pyraclostrobin WG showed control value of above 80% against Sclerotium rot caused by Sclerotium rolfsii except Pyribencarb SC. Also there was no chemical injury in reference amount and in fold amount respectively, compared to non treated control. Conclusion : From the above results, we selected four items of pesticides including Fludioxonil SC, Tebuconazole SC, Flutolanil EC, and Pyraclostrobin WG as effective chemicals against Sclerotium rot caused by Sclerotium rolfsii in Ixeridium dentatum.

Background : In the process of developing new disease-resistant cultivar of Rehmannia glutinosa, it is essential to screen plants based on resistance to their disease. But until now, we have relied on visual inspection for selection of disease-resistant cultivar. Therefore, this experiment was carried out to establish an efficient and reliable screening system and test the resistance to leaf spot disease in R. glutinosa cultivars developed until now. Methods and Results : We have tested 11 R. glutinosa cultivars developed so far. Rootstock of R. glutinosa were sown in 20 × 20 ㎝ plastic square port and grown in green house at 25 ± 5℃ for four months and then cutting leaves were inoculated with Phoma sp. HCRD 17103 by spraying spore suspention of fungus at concentration of 1 × 107 spore/㎖. The inoculated leaves were incubated in a dew chamber at 25℃ for 48h and then transferred to glass house (25 ± 5℃, RH 80% ≥ ). After 3 - 5 days when the most susceptible cultivar had lesion area over 40%, disease severity of the cultivars was investigated on a scale of 0 - 9 (0 = healthy, 1 = 0 - 1%, 3 = 1.1 - 10%, 5 = 10.1 - 20%, 7 = 20.1 - 40%, 9 = 40.1% over). The degree of resistance was determined according to the disease index (0 and 1 = resistant, 3 = moderately resistant, 5, 7 and 9 = susceptible). As a result of experiments according to the above criteria, the disease index of cultivar Gogang, Dagang, Segang, Yeongang, Wongang, and Dahwang was 1, cultivar Togang, Daegyung, Jiwhang 1 and Goryeo was 3, cultivar Hwanggang was 9, respectively. Conclusion : From the above results, six cultivars such as Gogang, Dagang, Segang, Yeongang, Wongang, and Dahwang were resistant, four cultivar such as Togang, Daegyung, Jiwhang 1 and Goryeo were moderately resistant, and only one cultivar Hwanggang was susceptible to the leaf spot disease by Phoma sp. This experiment used only leaves and we plan to use whole plants in the future to see more accurate resistance response.

Background : This study was conducted to investigate the production competitiveness of medicinal crop. The results of this study were intended to be used as basic data for establishing the direction of R&D needed for domestic medicinal crop farming system. Methods and Results : For data analysis, frequency, percentage and average and Chi square (× 2) value were used. The survey showed that the cultivated crops of the respondents were medicinal crop. Firstly, there was a significant difference in farming disability by farming career. The 'cultivation method (60%, 42.1%)' was high in farming preparer and beginner (≤ 3 years), but the disability in 'cultivation method' decreased as the farming career was longer. Respondents who had more than 10 years of farming career complained of 'climate problem (24.0%)' followed by 'cultivation method (20.0%)' and 'pest control (20.0%)'. There was also a significant difference in the farming disability by medicinal crops farming career. The 'cultivation method (50.0%)' was the highest of the farmers who had 1 year of medicinal crops farming career, however the respondents with more than 4-ears of career complained of 'climate problems (27.3%)' and 'pest control (23.6%)', but 'cultivation method' was low as 18.2%. Secondly, there was a difference in sales disability by farming career, and cross-sectional analysis was statistically significant at × 2 = 41.320. The respondents who were preparer for farming had the biggest sales disability at 'shortage of market (44.4%)', and the rates decreased gradually as the farming career increased. Respondents more than 10-ears had the biggest sales disability as ‘uncertain market price (50.0%)’, and 'shortage of market' was low as 12.5%. Cross-sectional analysis of sales disability by medicinal crops farming career showed that × 2 was 49.705, which was statistically significant. Farmers with no career in cultivating medicinal crops had the biggest sales disability at 'shortage of market (40.0%)' and farmers with more than 4 years of career complained of 'uncertain market price (42.2%)'. Lastly, there was a statistically significant difference in cultivation performance by medicinal crops farming career. The respondents with a career of less than 1 year had the highest proportion at 'medium (48.5%)', but those with more than 4-ears of career had the highest rate of 'creation of profit (43.1%)'. Conclusion : As farming career and medicinal crops farming career increased, environmental factors such as climate and pest problems affected in cultivation stage significantly, further uncertain market price gave a large factor in sales stage.