• Journal of Veterinary Science
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• 제19권6호
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• pp.788-797
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• 2018

In many countries, vaccines are used for the prevention of foot-and-mouth disease (FMD). However, because there is no protection against FMD immediately after vaccination, research and development on antiviral agents is being conducted to induce protection until immunological competence is produced. This study tested whether well-known chemicals used as RNA virus treatment agents had inhibitory effects on FMD viruses (FMDVs) and demonstrated that ribavirin showed antiviral effects against FMDV in vitro/in vivo. In addition, it was observed that combining the administration of the antiviral agents orally and complementary therapy with vaccines synergistically enhanced antiviral activity and preserved the survival rate and body weight in the experimental animals. Antiviral agents mixed with an adjuvant were inoculated intramuscularly along with the vaccines, thereby inhibiting virus replication after injection and verifying that it was possible to induce early protection against viral infection prior to immunity being achieved through the vaccine. Finally, pigs treated with antiviral agents and vaccines showed no clinical signs and had low virus excretion. Based on these results, it is expected that this combined approach could be a therapeutic and preventive treatment for early protection against FMD.

• Reproductive and Developmental Biology
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• 제38권3호
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• pp.115-121
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• 2014

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, $0.1{\mu}g/mL$, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by $0.01{\mu}g/mL$ compared with other groups (p< 0.05). Moreover, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.30-30
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• 2001

The culture of preantral follicles has important biotechnological implications through its potential to produce the large quantity of oocytes for embryo production, transgenesis research, conservation of rare breed, and a potential source of ovarian genetic material. The present study was conducted to establish the optimal conditions of in vitro culture for intact bovine preantral follicles; and to examine the developmental ability of oocytes derived from the in vitro-grown preantral follicles; and to investigate the effects of various concentrations of FSH and LH on these processes. Bovine preantral follicles (150 $\pm$ 1.2${\mu}{\textrm}{m}$), surrounded by theca cell, were isolated enzymetically and mechanically from ovarian cortical slides in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagens and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium with insulin, transferrin, and selenite. The survival was tested by frypan Blue and Hematoxylin. The survival and growth rates of follicles were higher in FSH treatment groups than these in control (P<0.001), but there were no significant differences between the LH treatment groups and the control. In 25 days, the survival and growth rates of follicles in FSH and LH treatment group (50%, 300$\pm$1.0${\mu}{\textrm}{m}$) were higher than in FSH treatment group (40%, 244$\pm$0.5${\mu}{\textrm}{m}$) and the control group (25%, 160$\pm$ 1.0${\mu}{\textrm}{m}$). Fifty-five percent of healthy antral follicles were obtained, and 60% of the oocytes complete meiotic maturation to the metaphase II stage. Twenty-two percent of the mature oocytes underwent cleavage, and 9% developed to the blastocyst stage. In this study, in vitro-grown oocytes (111 $\pm$ $1.5mutextrm{m}$), under our culture conditions, were not equivalent in size to the in vivo-grown oocytes (130$\pm$1.3${\mu}{\textrm}{m}$). Therefore, these results suggest that bovine preantral follicles with intact theca cell can grow to the antral stage in 25days, and that oocytes from those follicles can acquire the meiotic competence and normally undergo fertilization and development to the blastocyst stage. However, the developmental capacity of in vitro-grown oocytes is presumably not comparable to those of the in vivo counterparts.

• 한국수정란이식학회지
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• 제30권1호
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• pp.23-31
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• 2015

Catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa Libosch. It possesses a broad range of biological and pharmacological activity including anti-tumor, anti-inflammation and anti-oxidant by acting as a free radical scavenger. Therefore, in this study, the effects of catalpol on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in porcine embryos. After in vitro maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) supplemented with catalpol (0, 100, 200 and $400{\mu}M$, respectively). Blastocyst development not significantly improved in the catalpol treated group when compared with control group. Otherwise, the intracelluar levels of ROS were decreased and the numbers of apoptotic nuclei were reduced in the catalpol ($100{\mu}M$) treated porcine blastocysts (P<0.05). On the other hand, blastocyst development was significantly improved in the catalpol ($100{\mu}M$) treated group when compared with the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Otherwise, the intracellular levels of ROS in catalpol ($100{\mu}M$) treated group were significantly decreased in the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Furthermore, the total cell numbers of blastocysts were significantly increased (P<0.05) in the catalpol ($100{\mu}M$) treated group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress, whereas numbers of apoptoic nuclei were significantly reduced (P<0.05). In conclusion, our results indicate that treatment of catalpol may have important implications for improving developmental competence and preimplantation quality of porcine embryos through its anti-oxidant and anti-apoptotic effect.

• 한국수정란이식학회지
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• 제17권1호
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• pp.55-59
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• 2002

ICSI시 동결 융해한 부고환 정자의 이용 가능성을 알아보고자 난자의 배양시 체외성숙율과 활성화 처리를 한 난자와 동결 융해한 부고환 정자로 ICSI시 체외발생율을 조사하였으며, 결과를 요약하면 다음과 같다. 1. 난포란을 회수 후 24시간 배양하였을 때 배양 시간에 따른 GV, MI, M II로의 체외성숙율은 각각 7/60(11.7%), 5/60(8.3%), 48/60(80.0%)였고 30시간 배양 시간에 따른 GV, MI, M II로의 체외성숙율은 각각 3/60(5.0%), 4/60(6.7%), 53/60(88.3%)였고 퇴화란은 각각 2/60(3.3%)와 1/60(1.7%)였다. 2. 동결 융해한 부고환 정자를 이용하여 활성화 처리를 한 난자에 ICSI를 하였을 때 상실배와 배반포로의 체외발생율은 각각 12/46(26.1%), 22/46 (47.8%)로서 비활성화처리 난자군 5/39 (12.8%), 10/39(25.6%)에 비해 높은 체외발생율을 나타냈다. 3. 활성화 처리를 한 난자에 신선정자, 부고환 정자 및 동결 융해한 부고환 정자로 ICSI시 체외 발생율은 각각 24/45(53.3%), 15/40(37.50%), l1/43 (25.6%)로서 신선정자에 비해 동결 융해한 부고환 정자처리군은 체외발생율은 약간 낮았지만 이용 가능성이 있음을 확인하였다.

• 한국가축번식학회지
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• 제24권1호
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• pp.69-76
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• 2000

수정란이식에 필요한 다량의 수정란을 생산하는 수단인 배양액과 적정 수정란의 수는 수정란의 체외발달에 많은 영향을 미치므로 이들 관계를 조사하여 수정란의 체외배양체계를 확립하고자 본 연구를 실시하였다. 도축장에서 채취한 난소에서 미성숙 난자를 채란하여 10% FBS가 첨가된 TCM -199 에 LH(10 $\mu\textrm{g}$/$m\ell$), FSH(35 $\mu\textrm{g}$/$m\ell$), estradiol-17 $\beta$(1 $\mu\textrm{g}$/$m\ell$)가 첨가된 체외성숙배양액에서 24시간 동안 배양 후, 동결정액은 Percoll-density gradients(45 vs. 90%)을 이용하여 700 g 에서 30분 동안 처리한 다음, 체외수정배양액 (IVF-Fert)에서 체외수정을 유도하였다. 수정이 확인된 수정란은 50 ${\mu}\ell$ 배양액에 1, 25 또는 50개의 수정란을 난구세포와 공배양을 하여 9일 동안 배양하였다. 일정한 배양액에서 수정란의 수가 체외수정란의 발달에 미치는 요인을 분석하고, 개별배양 또는 그룹배양 시 난구세포와의 공동배양 효과를 조사한 결과는 다음과 같다. 1. 50 ${\mu}\ell$ 배양액에 1개, 25 또는 50개의 수정란을 수정 후 9일 동안 배양한 결과, 25와 50개의 수정을 배양했을 경우에는 발달율이 36.5 와 26.5%를 보여 1개의 수정란을 배양했을 경우에서의 발달율 6.2% 보다 유의적으로 높은 발달율을 얻었다(P＜0.05). 2. 1, 25, 50개의 수정란을 배양시 수정 후 6일째 발달율은 1.0~3.5%였으나, 수정 후 7, 8, 9 일째의 발달율은 1개의 수정란을 배양하는 것보다 25, 50개의 수정란을 배양한 처리구에서 유의적으로 높은 발달율을 얻었다 (P<0.05). 3. 일정한 배양액에서 1, 25 및 50개의 수정란을 배양 시 수정 후 8일째의 배반포 수정란의 세포수를 조사한 결과, 1개의 수정란을 배양시 배반포 수정란의 수는 93.0개였으나, 25, 50개의 수정란을 배양시는 각각 112개의 세포수를 얻어 유의적으로 높은 세포수를 나타내었다 (p＜0.01). 4. 일정한 배양액에 1개 또는 25개의 수정란을 난구세포와 공배양을 하거나, 하지 않았을 때의 발달율은 각각 15.0와 3.7% 또는 34.5와 9.0%로서 난구세포와 공배양을 하는 것이 유의적으로 높은 발달율을 나타내었다 (p＜0.01 수정 후 8 일째의 배반포 수정란의 세포수도 각각 96.1와 82.0개 또는 116.4 와 96.5개로서 난구세포와 공배양을 한 처리구에서 유의적으로 높은 세포수를 나타내었다 (p＜0.01). 이상의 결과를 요약하면, 수정란의 수가 체외수정란의 체외발달에 중요한 영향을 미친다는 것을 알 수 있었으며, 다량의 체외수정란을 생산하여 수정란 이식에 이용하기 위해서는 체외성숙 / 수정된 체외수정란을 일정한 배양액 (50${\mu}\ell$) 에 25, 50개의 수정란을 난구세포와 공배양하는 것이 높은 배 발달율과 세포 수를 얻 을 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제30권4호
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• pp.333-340
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• 2003

Objective: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). Materials and Methods: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: $32.4{\pm}3.8$ years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10, 000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in $10{\mu}l$ YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. Results: Th e mean number of oocytes cultured in the IVM/IVF cycles was $24.7{\pm}10.6$. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. Conclusion: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.

• 한국수정란이식학회지
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• 제24권2호
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, 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 cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제23권6호
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• pp.806-813
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• 2010

Animal bioreactors have been regarded as alternative tools for the production of limited human therapeutic proteins. The mammary glands of cattle are optimal tissues to produce therapeutic proteins that cannot be produced in large amounts in traditional systems based on microorganisms and eukaryotic cells. In this study, two knock-in vectors, pBCTPOKI-6 and pBCTPOKI-10, which target the hTPO gene on the bovine beta-casein locus, were designed to develop cloned transgenic cattle. The pBCTPOKI-6 and pBCTPOKI-10 vectors expressed hTPO protein in culture medium at a concentration of 774 pg/ml and 1,867 pg/ml, respectively. Successfully, two targeted cell clones were obtained from the bovine fibroblasts transfected with the pBCTPOKI-6 vector. Cloned embryos reconstructed with the targeted nuclei showed a lower in vitro developmental competence than those with the wild-type nuclei. After transfer of the cloned embryos into recipients, 7 pregnancies were detected at 40 to 60 days of gestation, but failed to develop to term. The results are the first trial for targeting of a human gene on the bovine milk protein gene locus, providing the potential for a large-scale production of therapeutic proteins in the animal bioreactor system.

• 한국초지조사료학회지
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• 제38권4호
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• pp.320-324
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• 2018

본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).