Earticle

Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.

사람의 경우, 간은 성별에 의존적으로 다른 크기와 분비되는 생리 대사물질의 양이 다 른 것으로 알려져 있지만, 대부분의 연구는 성별의 영향이 고려되지 않고 있다. 따라서 이 연구에서는 성별에 따른 간의 기능을 규명하기 위해 같은 주령의 암수 마우스(11주령, C57BL/6J, 5쌍)의 간을 사용하여 생리대사 기능을 평가하고, 암수의 체세포유래 교차분 화 유도간세포(induced Hepatocyte)를 만들어 성별에 따른 생리 대사기능이 유지되는지를 확인하였다. 특히 마우스의 약물독성 대사에 주요한 공통발현유전자(Cyp3a11와 Cyp3a25) 와 성 특이적 CYP enzyme 유전자들(Cyp2d9, Cyp8b1, Cyp2a4, Cyp3a41)의 mRNA 발 현을 확인하였다. 비록 개체 간의 차이는 존재하였지만, 공통적으로 발현되는 Cyp3a11는 웅성에서 증가를 보였고, Cyp2d9, Cyp8b1는 웅성에서 Cyp2a4, Cyp3a41는 자성에서 증 가가 확인되었다. 추가적으로 같은 연령의 암놈의 태아들에서 10개의 mouse embryonic fibroblast을 분리배양하고 성별을 확인하였다. 이중 암수 각1개의 fibroblast를 선택하고 lentivirus를 이용하여 3개의 간 전사인자 (hHNF1A, hHNF4A, hFOXA3)가 각각 삽입된 벡 터를 감염시키고, 6주 동안 간 분화 배양액에서 분화를 유도하였다. Cyp 유전자 중 공통 발현 유전자인 Cyp3a11의 경우, 간의 결과와 동일하게 웅성에서 자성보다 증가됨이 확 인되었다. 이상의 결과들에서 볼 때, 성별 따른 간 특성은 교차분화된 암수 유도간세포에도 일부 유지됨을 확인할 수 있었다. 따라서 세포치료제나 신약개발을 위한 독성 평가용 세포주 도 성별 맞춤형 구축이 필요함을 시사한다.

Spermatogonial stem cells (SSCs) are expected to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for transgenic animal technology. We have established an efficient isolating for SSCs from 1-day-old chicken testicular cells. This present study is aimed to carry out the culture and characterization of chicken SSCs. Disassociation of chicken SSCs were performed using two-step enzymatic digestion of type 4 collagenase and trypsin and were seeded in a twelve-well plate. In primary culture was observed colony forming on day four post incubation. In the subsequent passages, chicken SSCs were positive for alkaline phosphatase activity and showed positive expression for the transcription factor, Oct 4. Immunoblotting analysis revealed that the chicken SSCs showed expression for the GRP78. Therefore, our findings may have implications for developmental biology and regenerative medicine by using chicken as the transgenic animal model.

Membrane cofactor protein(MCP)은 세포막에서 발현하는 보체 조절 단백질로서, 보체 부산물인 c3b, c4b 등을 불활성화시킴으로써 항원-항체 반응과 염증반응을 억제하는 역 할을 한다. Thrombomodulin(TBM)은 protein C를 활성화시켜 혈액응고를 억제시키는 기 능을 수행하는 단백질로서 높은 수준으로 발현되면 번식장애를 유도한다고 알려져 있다. 그런데, MCP cDNA를 이용한 발현벡터를 세포에 도입하면 mRNA 수준에서는 효율적으 로 과발현이 유도되나, 단백질로 발현되는 수준이 낮아, MCP는 과발현 유도가 어려운 유전자로 알려져 있다. 본 연구에서는 MCP cDNA의 전사체로부터 번역되는 효율이 낮아 단백질 발현 수준이 낮은 것으로 판단하여, 아미노산 서열의 변경은 없이 cDNA 유전자 서열을 치환, 변경하면 단백질 발현 수준이 증가하는지 분석하였으며, 동시에 TBM 발현 수준을 연결한 프로모터로 조절이 가능한지 분석하였다. 이를 위해 CAG 프로모터에 염 기서열을 치환한 MCP cDNA와 2A 시스템을 활용한 TBM cDNA를 연결한 발현 벡터 (CAG-pmMCP_2A_TBM)와 CAG 프로모터에 염기서열을 치환한 MCP cDNA와 돼지 Icam- 2 프로모터에 TBM cDNA를 연결한 발현벡터(CAGpmMCP-pI2TBM)를 각각 제작한 후 porcine ear fibroblast(pEF), porcine aorta endothelial cell(pAEC)에 도입하였다. pEF 세 포와 pAEC에서 CAG-pmMCP_2A_TBM 벡터는 대조군에 Flow cytometry로 분석한 단백 질 수준에서는 염기서열 치환에 의해 MCP 발현이 유의적으로 증가하는 것을 확인하였 고, pAEC에서 TBM 발현 수준은 돼지 Icam2 프로모터가 CAG 프로모터보다 높아 프로 모터의 선정으로 발현 수준을 조절할 수 있다는 것을 확인하였다. 단백질로 과발현된 MCP가 보체 활성화 억제 기능을 수행하는 지 알아보기 위하여, 발현벡터가 도입된 pEF 세포에 정상 cynomolgous monkey 혈청을 첨가하여 cell proliferation assay를 수행한 결 과 세포 사멸이 유의적으로 감소하는 것을 확인하였다. 이러한 결과는 MCP cDNA의 염 기, 서열 변경을 통해 보체 활성 억제 기능을 효율적으로 수행하는 단백질의 과발현을 유도할 수 있으며, 프로모터의 선정으로 세포나 동물의 생리에 유해한 해를 끼치는 유전 자의 발현 수준 조절이 가능하다는 것을 보여준다.

대기온도의 상승은 번식효율을 비롯하여 가축 생산성에 영향을 미치게 된다. 특히 고온 스트레스에 대응하여 항상성유지를 위해 Heast shock factors (HSFs)을 활성화를 통해 Heat shock protein (HSPs)를 발현하게 되어, 세포의 기능, 구조, 세포 사명 등을 결정하 게 된다. 특히 HSP70 faimly 유전자 (HSPA) 등은 특별히 중요한 표지 인자로 쓰이고 있 다. 또한 고온 스트레스는 산화스트레스를 통해 ROS를 생산 미토콘드리아 손상 등을 일 으키게 되며, 이에 대응하기 위해 superoxide dismutase, catalase, gultahtione, peroxidase, peroxidredoxin 등이 사용되어 독성을 해결한다. 본 연구는 고온 스트레스 하에서 암컷 생식기인 난소내에서의 Hsp와 Prdx의 반응을 제브리피쉬를 통해 수행하였다. 정상 수온인 28.5℃에서 34℃로 높여 2시간 키운 후, 정상온도로 낮추어 48시간 키운 후, Tricane을 이용 정상, 고온, 회복 각 3단계에서 난소를 채취하여 qRT-PCR을 수행하였다. Hsp70l, Hspa1a, Hspa1b, hspa13은 유의성 있게 증가하였으나, Hspb1은 고온 스트레스, 회복 모두 큰 차이 없었다. 반면에 Hsp70l은 고온하 발현양도 크게 증가하고, 회복시 급 속히 감소하였다. Prdx1-6는 고온 하에서 크게 변하지 않았으나, 오히려 회복시 모든 Prdx1-6 발현량이 증가하였다. 본 연구는 모델 동물을 이용하여 고온 스트레스 하에 난 소에서 반응하는 두 유전자군의 발현 양상을 분석하였으며, 한우 등 중소가축에 대한 실 험을 위한 기초 자료로써 큰 의미가 있다.

Inhibition of MEK1/2 and GSK-3(2i system) is useful for maintenance of navie state of embryonic stem cells in various species. However, the role of 2i system in porcine preimplantation stage embryos is not clear. In this study, we investigated the quality of blastocyst and pluripotent-related factors by 2i system and epigenetic modification status by 2i system during porcine early embryo development. Treatment of MEK1/2 inhibitor PD0325901 (4 uM), GSK-3 inhibitor CHIR99021 (0.3 uM) and 2i (PD0325901+ CHIR99021) did not enhanced the rate of blastocyst formation. However, the size of blastocyst was increased in 2i treated embryos. TUNEL assay showed that CHIR- 99021 and 2i treatment enhanced blastocyst quality. Furthermore, 2i treated blastocyst increased OCT4 and SOX2 positive cells and decreased GATA4 positive cells. Methylation of histone H3 lysin trime3 (h3k9me3) were reduced, and histone H3 deacetylaton (h3k9ac) were increased in 2i treated embryos. Furthermore, bi-sulfite sequencing results showed that 2i treatment groups demethylated in satellite I region. And, RNA methylatransferase protein METTL3 was increased while demethylase FTO was increased after treatment of 2i. In conclusion, our findings strongly suggest that 2i system can be useful method for increasing of blastocyst quality by affecting of ICM/TE formation by regulating epigenetic modification during early embryo development.

DNA repair protein Rad51 homolog 1 (Rad51) plays a central role in Homologous recombination (HR) repair. HR depends on the formation of a Rad51 recombinase filament that facilitates strand invasion. However, whether Rad51 influences the porcine oocytes during maturation is unknown. The objective of this study is to investigate the expression and function of Rad51 during porcine oocytes maturing in vitro. Rad51 was mainly localized at the nuclear in the germinal vesicle (GV) stage and widely distributed in the cytoplasm form GV breakdown (GVBD) to MII stage. DNA damage inducing by the Etoposide was identified by the Rad51 foci formation which was localized with γH2AX. Inhibition of Rad51 increased the DNA damage, and induced metaphase I arrest with spindle defects, chromosomal misalignments and abnormal spindle assembly checkpoint (SAC) activity. Inhibition of Rad51 also increase ROS level and induced the abnormal mitochondrial distribution. Our results indicate that Rad51 play a critical role to maintain chromosome integrity as well as mitochondria activity during the porcine oocyte maturation.

CPEB-mediated tight junction assembly was identified in mammary epithelial cell polarity but not known in early embryos. The objective of this study is to determine expression and function of CPEB2 during blastocyst formation in porcine parthenote developing. Transcriptional level of CPEB was identified by qPCR, and protein expression was observed by immunocytochemistry. Double strand RNA(dsRNA) of CPEB2 was injected into zygotes to identify and cultured in vitro to identify the function of CPEB2. The formation of tight junction protein was confirmed by FITC dextran assay. The overall localization of CPEB3 and cell junction related proteins (ZO-1, CAR, Occludin, E-cadherin, ß-catenin) were also confirmed in CPEB2 knock downed embryos. CPEB2 proteins were identified in nucleus until 8-cell stage, but localized in cytoplasm in morula and blastocyst embryos. Knock down of CPEB2 using double strand RNA reduced development to blastocysts and blastocyst size. FITC-dextran assay showed that GFP-dextran positive embryo was increased in the KD (28.3%) than the control (5.7%). ZO-1, CAR, and OCLN, was mislocalized in CPEB2 KD embryos. These results suggest that the formation of tight junction protein by CPEB2 plays a crucial role in embryonic development in mammals.

Dynamic changes in the actin network are crucial for the cortical migration of spindle and establishment of polarity to ensure asymmetric division during meiotic maturation. Here, we show that Filamin A (FLNA) is an essential actin regulator that controls spindle migration and asymmetric division during oocyte meiosis. FLNA was localized in the cytoplasm and enriched at the cortex and near the chromosomes. Knockdown of FLNA impaired meiotic asymmetric division and spindle migration with a decrease in the amount of cytoplasmic actin mesh and cortical actin levels. Moreover, FLNA knockdown reduced the phosphorylation of Cofilin and Rho kinase (ROCK) near the spindle. Similar phenotypes such as decreased filament actin levels, impaired spindle migration and polar body extrusion were observed when active Cofilin (S3A) was overexpressed or ROCK was inhibited. Notably, we found that FLNA and ROCK interacted directly in mouse oocytes. Taken together, our results show that FLNA plays crucial roles in asymmetric division during meiotic maturation by regulating ROCK-Cofilin-mediated actin reorganization.

Mammalian oocytes lack a centriole that acts as a microtubule organization center (MTOC) in most somatic cells. During oocyte maturation, MTOCs undergo remodeling process including decondensation, fragmentation and self-organization. But the underlined mechanisms of MTOC remodeling in mouse oocyte are not well understood. We showed that two pericentriolar proteins, Cep192 and Cep152 play crucial roles of MTOC remodeling. Cep192 is present in MTOC at all stages of oocyte maturation, and the depletion of Cep192 induces ablation of MTOC, delay inspindle formation and abnormal chromosomal alignment in spindles. In the case of Cep152, Cep152 localization of MTOC is limited at the germinal vesicle (GV) stage and then disappeare from the MTOC after germinal vesicle breakdown (GVBD). Cep152 exclusion from MTOC is involved in the fragmentation of MTOC and it is regulated by CDK1 activity. Together, our results demonstrate the different roles of Cep192 and Cep152 in MTOC remodeling and novel regulatory mechanism during meiotic spindle formation in mouse oocyte.

Gonadotropin receptors are members of the seven transmembrane receptor families. Several point mutations in the third intracellular loop have been identified in the follicle stimulating hormone receptor (FSHR) gene leading to constitutive activation and inactivation of the receptor. The FSHR gene mutations cause a form of ovarian resistance (savage syndrome) in females and small testes and abnormal sperm parameters in males. In eelFSHR, we generated 1 type of constitutive activating mutation (D566G) and 2 types of constitutive inactivating mutations (A189V, N191I) to investigate how they work on hormone-receptor interaction and receptor activation system on eel rec- FSH. To assess the functional effects of 3 receptor mutations directly, wild type and mutant eelFSHRs were constructed to pVSV-G expression vector and transiently transfected to CHO-K1 cells with expressing vector cloned in pVSVG (6 well/plate; 2,500 ng). And then cAMP accumulation by rec-eelFSH stimulated was measured. Rec-eel- FSH produced a concentration-dependent increase in cAMP production in wild-type receptor. Next, we transfected with D566G plasmid (6 well/plate; 5,000 ng). It was drastically elevated basal cAMP level compared to wild-type. The basal cAMP levels were 11.5 nM and 25.8 nM, respectively. Thus, D556G in the basal cAMP responsiveness was 2.2 times than that of wild type. However, the inactivation mutant A189V did not obviously altered. In summary, we suggest that mutation of D566G is responsible for constitutive activation in eel FSHR like mammalian FSHR.

Gonadotropin receptors are members of the seven transmembrane (TM) receptor families. Several point mutations in TM II, III, V and VI have been identified in the luteinizing hormone receptor (LHR) gene, leading to constitutive activation and inactivation of the receptor. In eelLHR, we generated 3 types of constitutive activating mutations (M410T, L469R and D590Y) and 2 types of constitutive inactivating mutations (D383N and Y546F) to investigate how they work on hormone-receptor interaction and receptor activation system on eel. To assess the functional effects of 5 receptor mutations directly, wt and mutant eel- LHRs were transiently expressed in CHO-K1 cells, and basal and recombinant eel LH-stimulated cAMP and IP-1 accumulations were measured. Rec-eelLH (0.076～1,200 ng/mL) produced a concentration-dependent increase in cAMP production in wt eelLHR expressing cells with an EC50 of 160 ng/mL and basal cAMP level of 2.6 nM. In contrast, the L469R activation mutant had most elevated (16.88 fold higher than wt) basal cAMP production (basal cAMP level=43.9 nM). Compared with the wt eelLHR, all the activation mutant receptors produced higher basal levels of cAMP (18.4 nM for D590Y and 7.9 nM for M410T). However, eelLH-stimulated (0.076～1,200 ng/mL) basal cAMP levels in the constitutive inactivation mutants D383N and Y546F did not obviously altered from that in wt eelLHR. D383N mutation increased the EC50 value to 185 ng/mL (inhibited receptor activity to 86%), while Y546F mutation increased that to 170 ng/mL which implies that receptor activity was inhibited to 94% only. As seen in IC50 values in IP-1 accumulation, activity for M410T mutant was 19% higher than that for wt receptor, but other activation mutants did not show any difference in IP-1 production. In case of inactivation mutants, there were no significant differences in IP-1 production and only 7% decreased activity was identified in D383N mutant. In summary, we have demonstrated 3 mutations that are responsible for constitutive activation of eelLHR. Although predicted 2 inactivation mutations led to slightly diminished activation of receptor, those could not impair signal transduction of eelLHR.

Equine-chorionic gonadotropin (eCG) is a member of the glycoprotein family with LH, FSH and TSH. In non-equid species, eCG shows both LH- and FSH-like activities except the horses and has a high affinity for both FSH and LH receptors. These receptors were synthesized in granulosa/theca and Sertoli/Leydig cells in ovary/testis and tansported to the membrane surface. Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. The LH/CGR is a member of the family of G-protein-coupled receptors that the receptor was composed of a single polypeptide chain. To assess the functional effect of the D576Y mutation in the eLH/CGR, wild type and mutated equine LH/CGR were transiently expressed in CHO-K1 cells and cAMP accumulation was measured. rec-eCG produced a concentration-dependent increase in cAMP production in cells expressing the wild-type eLH/CGR, with an EC50 of 21.3 ng /mL and mean maximal stimulation (170.1±11.9 nM). However, the mutant eLH/CGR -D576Y produced a 6.4-fold increase in basal cAMP production in CHO-K1 cells, indicating that it was constitutively active. In the inactivating mutant (R464H), cAMP responsiveness was completely flat. But cAMP concentration for activating m mutant (M- 398T) was almost same pattern as wild-type receptor. Thus, our result suggest that an inherited disease FMPP is due to a constitutively activating mutations in a G-protein coupled hormone receptor.

Progesterone primes the endometrium for possible implantation and inhibits uterine contractions until birth. 20α-Hydroxysteroid dehydrogenase (20α-HSD) is a member of the aldo-keto reductase (AKR) family. These enzymes play pivotal roles in the modulation and regulation of steroid hormones, such as androgens, estrogens, and progestins, and are thus considered important targets for drug design. In the present study, we investigated the expression pattern of 20α-HSD during pregnancy in bovine luteal tissues. And, the amounts of mRNA were determined in cultured cells of the corpus luteum cells(CL). The localization of 20α-HSD was also determined in luteum during early pregnancy. Analysis of expression pattern of progesterone inhibitor gene 20a-HSD and apoptosis mechanism in the culture cell. Collected of during cells were the cultured under 20a-HSD(0.375 ug/mL(type 1), 0.75 ug/mL(type 2), and 1.125 ug/mL(type 3) in 48 hr. and to determine the function of 20α-HSD gene during pregnancy, we collected the pregnant corpus luteal on 30, 60 and 90 days of pregnancy. The first results found that the level of 20α-HSD was the most highly expressed in the corpus luteal day 90. And the luteum progressively increased from day 30, 60 to 90. Furthermore apoptotic detected was the most highly expressed in the corpus luteal day 30 and 90. The secondly results, that the type 1 cultured, 20α-HSD and Casp-3 protein was the most highly expressed throughout cytoplasm. However, decreased 20α-HSD expression on the type 3. Interestingly, 20α-HSD and Casp-3 proteins was lowest expressed in type 2, cytosol is showing levels higher than cytoplasm. These results suggest that the 20α-HSD plays a role in maintaining normal pregnancy at least partially by controlling the progesterone hormone concentrations during development of copus luteum cell.

본 연구는 돼지의 번식 효율을 개선하고자 발정동기화 방법에 따른 예정시각 인공수정 이 수태율에 미치는 영향을 조사하고자 하였다. 돼지의 발정동기화를 위하여 KNP(Korean Native Pig) 및 요크셔 경산돈 각각 6두에 대해서 급이식은 REGUMATEⓇ(altrenogeste 20 mg/5 mL, dose, Hoechst Roussel Vet., France)를 1일 1회 오전 08:00시에 5 mL씩 10일간 급여하고, 삽입식은 Ring-CIDR (Ring type CIDR, 0.4 g progesterone pre device, 4-Pregnene-3,20-dione, SIGMA, USA)를 10일간 질 내에 삽입시킨 후, 제거 또는 급여 중단한 후 두당 PGF2⍺(Lutalyse, PHARMACIA, Belgium) 1.5 mL 및 PG600(PMSG 400 IU+hCG 200 IU) 2.5 mL를 각각 주사하였으며, 36시간 후 배란을 촉진하기 위하여 2.0 mL GnRH(Fertagyl, ganadolelin 100 mg/mL, Boxmeer, Holanda)를 주사하고, 6시간 및 12시간 후에 각각 인공수정하였다. 처리별 각각 발정발현율은 50% 및 66.7%를 나타 냈으며, 수태율은 각각 16.7% 및 33.3%를 나타내었다. 수태율은 인공수정 후 45일경에 초음파진단기(SA600A, 5.0Mz, ㈜메디슨, 한국)에 의해서 조사하였다. 경산돈의 발정동기화 방법으로 Ring-CIDR의 질내 삽입법이 급이식보다 발정발현율 및 수태율이 다소 높았다. 질 내 삽입법의 경우, 6개체 중 2개체에서 삽입 후 2일 이내에 탈 락되어 재 삽입하였으며, 급이식의 경우에는 초기에 아주 드물게 2～3개체가 사료를 먹 지 않고 남긴 사례가 발생하였다. 발정 징후는 다소 미약하였으나, 인공수정은 발정 발현 에 관계없이 예정된 시간에 실기하였다. 예정시각 인공수정율이 자연종부에 비해 낮게 나타난 것은 발정동기화 처리 반응이 각각 낮게 발현된 것에 기인된 것으로 사료되었다.

1990년대 이후로 농촌지도기관의 경영지도의 개념이 컨설팅으로 전환되었다. 지도는 일방적으로 지식이나 기술을 전달하는 것인 반면 컨설팅은 상호 정보교환을 통하여 진단 후 처방하는 방법이다. 이러한 방식은 농산물시장이 국제화되면서 경쟁력을 갖추기 위하 여 필요하고 농가 경영환경이 다양화 되면서 농가별 맞춤식 경영개선을 위하여 필요하게 되었다. 이러한 흐름에 맞추어 2000년 초까지 농촌진흥청은 60개 품목에 대하여 표준 경 영진단표를 개발하였다. 하지만 상대적으로 비중이 적은 작목에 대한 진단표가 개발되지 않아 본 연구를 수행하게 되었다. 본 연구는 기능성이 우수하여 신소득 작목으로 주목받 고 있는 흑염소를 상대로 하였다. 본 연구를 수행하기 위하여 1차적으로 전문가 협의회 를 실시하였다. 분야별 전문가 의견을 수렴하여 경영성과지표 3개 항목과 진단항목 4개 요소를 설정하였다. 경영성과지표 3개는 자양육성율, 분만간격, 사육두수이고, 진단항목 4 개 요소는 번식후보 흑염소 선발, 개체관리, 사육시설, 경영관리 4개이다. 이렇게 경영성 과지표와 진단요소를 설정한 후 2차적으로 전문가 검증과 가중치 설정을 위하여 델파이 조사를 2차에 걸쳐 수행하였다. 델파이 조사를 위하여 흑염소 관련 전문가를 선정하였다. 최대한 객관성을 확보하기 위하여 개별 이메일 설문조사를 실시하였다. 1차 델파이 조사 결과 경영성과지표의 사육두수 항목 중 3단계와 4단계에서 CV-value의 값이 50%를 상회 하여 재조사를 실시하였다. 그리고 세부구성유소에서는 번식후보 흑염소 선발은 모두 일 치하였고 개체관리의 세부요소 예방접종 및 구충, 흑염소 폐사율, 사료급여에서 리커트 척도(1∼5점) IQR 값이 1을 넘어 전문가간 불일치를 보여 재조사를 실시하였다. 그리고 진단표 4개 항목에 대한 전문가 가중치 의견 수렴결과 번식후보 흑염소 선발 25%, 개체 관리 35%, 사육시설 20%, 경영관리 20%로 설정하였다. 1차 델파이 조사결과, 불일치로 나온 항목은 통계적으로 제시한 값을 2차 조사시 다시 보여주고 일치여부를 설문하였다. 이 결과 경영성과지표의 사육두수 3단계와 4단계는 100% 일치를 보여 설정하였다. 그리 고 진단항목 개체관리의 세부요소 예방접종 및 구충, 흑염소 폐사율, 사료급여는 전문가 의 90% 제시값에 동의를 하여 설정하였다. 그리고 번식후보 번식방법도 90% 전문가 일 치로 설정되었다. 최종적으로 흑염소 경영진단표는 경영성과지표의 3가지 항목 자양육성 율, 분만간격, 사육두수를 각각 5단계로 설정하였고 진단항목은 번식후보흑염소 선발의 2 개, 개체관리의 10개, 사육시설 4개, 경영관리 6개 총 22개 세부요소를 설정하였다. 기 만들어진 경영진단표는 100농가의 실증 진단을 통하여 활용 가능성을 검토하였고 시군농 업기술센터에 보내져 전문축산지도사에 의하여 컨설팅 일환으로 활용된다.

Plasminogen activators system (PAs) is one of important physiological system for reproductive events such as oocyte maturation, acrosome reaction, fertilization, and uterine remodeling. It is consist of two-types of PAs (urokinase-type, uPA; tissue-type, tPA), uPA-specific receptor (uPAR), and type-1 PA inhibitor (PAI-1). In various type of cells, activity of these system was regulated by gonadotropin, cytokines, and hormones, however, regulation of mRNA expression in porcine uterine cells was not clear. Therefore, this study aimed to investigate the effect of progesterone (P4) on mRNA expression of uPA, tPA, uPAR, and PAI-1 in epithelial cells of porcine uterine endometrium. Epithelial cells were isolated from luminal epithelium in endometrium and cultured at 80% confluence. Then, it were subsequently incubated with different concentration of P4 (0, 0.1, 1, 5, 10, and 20 ng/mL) for 5 min and 24 h at 38.5℃, 5% CO2 in air. mRNA expression was measured by reverse-transcription PCR method. In results, expression of uPA mRNA in P4-exposed epithelial cell for 5 min was increased by 0.1 and 20 ng/mL P4 treatment. However, 1, 5, and 10 ng/mL P4 reduced uPA mRNA expression compared to 0.1 ng/mL treatment (p<0.05). On the other hand, all P4 treatment groups for 24 h enhanced mRNA expression of uPA. tPA mRNA in cultrued cells with P4 for 5 min and 24 h was enhanced by 10 and 20 ng/mL treatment, whereas 5 ng/mL P4 reduced. And 20 ng/mL P4 treatment increased uPAR mRNA expression in epithelial cells that were incubated for 5 min and 24 h. Similar to result of tPA mRNA, PAI-1 expression was enhanced by 5, 10, and 20 ng/mL treatment groups. The findings in this study show that mRNA expression of PAs system components were differently regulated by P4 in porcine uterine cells and further study regarding to regulation of this system was needed.

For the establishment of successful pregnancy the maternal immune system must tolerate the implanting semi-allogenic conceptus, but the mechanism underlying this process is not fully understood in pigs. Among many factors, members of the tumor necrosis factor superfamily (TNFSF) have been considered as important immune regulators in cell-mediated immunity. TNFSF members bind to their responsive receptor containing cytoplasmic death domain to induce apoptosis in immune cells. Action of TNFSF members at the maternal-fetal interface during pregnancy has been studied in some species including humans, mice and cows, suggesting that apoptosis of activated maternal immune cells in the uterine endometrium is a defense mechanism against rejection of semi-allogenic fetus. However, the expression and function of TNFSF members at the maternal-conceptus interface in pigs have not fully understood. Thus, to initiate the study on the role of TNFSF members for the establishment of pregnancy, we determined the expression of CD40 ligand (CD40LG), FAS ligand (FASLG) and TNFrelated apoptosis inducing ligand (TRAIL; TNFSF10) in the uterine endometrium and conceptus tissues during pregnancy in pigs. We obtained endometrial tissues from day (D) 12 and D15 of the estrous cycle, and D12, D15, D30, D60, D90, and D114 of pregnancy, conceptus tissues on D12, D15, and chorioallantoic tissues on D30, D60, D90, and D114 of pregnancy in pigs. Real-time RT-PCR analysis showed that CD- 40LG, FASLG and TNFSF10 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of CD40LG, FASLG and TNFSF10 mRNA in endometrial tissues were significantly increased on D15 of pregnancy and levels of FASLG and TNFSF10 were also high on D60 of pregnancy and decreased thereafter. Chorioallantoic tissues also expressed CD40LG, FASLG and TNFSF10 mRNA during mid- to late pregnancy. Immunohistochemical analysis showed that CD40LG and TNFSF10 proteins were mainly localized to luminal epithelial (LE) cells on D15 of pregnancy and amniotic membrane during late pregnancy, while FASLG protein was localized to LE cells during D30 to term and glandular epithelial cells during the estrous cycle and pregnancy. To understand the regulatory mechanism of endometrial CD- 40LG, FASLG and TNFSF10 expression by interferon gamma (IFNG), which is pro- duced by the implanting conceptus, we treated endometrial tissues with increasing doses of IFNG and found that IFNG increased the expression of FASLG and TNFSF10 mRNA, but not CD40LG mRNA. These results indicate that CD40LG, FASLG and TNFSF10 are expressed in a cell-type and stage-specific fashion in the uterine endometrium and chorioallantoic tissues during pregnancy, suggesting that CD40LG, FASLG and TNFSF10 may play an important role in the establishment of pregnancy by regulating the maternal immune response at the maternal-conceptus interface in pigs.

Uterine natural killer (uNK) cells are phenotypically and functionally specialized natural killer (NK) cells in the innate immune system. uNK cells are present at a high frequency in the uterine endometrium in humans and mice and have been shown to play a role both in regulating trophoblast invasion and in spiral artery remodeling. However, the function of uNK cells has not been well studied in pigs. Thus, to initiate the study on the role of uNK cells at the maternal-conceptus interface in pigs, this study determined the expression of several NK cell markers, including natural cytotoxicity triggering receptor 1 (NCR1), Fc fragment of IgG receptor III (FCGR3), neural cell adhesion molecule 1 (NCAM1), perforin 1 (PRF1) and granzyme B (GZMB) in the uterine endometrium during the estrous cycle and pregnancy in pigs. We obtained the endometrial tissues from gilts on day (D) 0, D3, D6, D9, D12, D15 and D18 of the estrous cycle and on D12, D15, D30, D60, D90 and D114 of pregnancy, and chorioallantoic tissues on D30, D60, D90 and D114 of pregnancy. Real-time RT-PCR analysis showed that the expression of NCR1, FCGR3, NCAM1, PRF1 and GZMB mRNAs was detected in the uterine endometrial tissues. Levels of NCR1, FCGR3, PRF1 and GZMB mRNAs in the uterine endometrium were highest on D15 of pregnancy. Levels of NCR1 mRNA were decreased toward term pregnancy. FCGR3 mRNA levels were constant during mid pregnancy. Expression levels of PRF1 and GZMB mRNA during pregnancy showed a biphasic pattern with the highest levels on D15 and D60 of pregnancy. However, levels of NCAM1 mRNA were highest on D12 of the estrous cycle. RT-PCR analysis showed that NCAM1 mRNA, but not NCR1, FCGR3, PRF1 and GZMB mRNAs, was detectable in conceptus on D12 and D15 of pregnancy. Chorioallantoic tissues also expressed NK cell markers, except NCR1, with increasing levels of FCGR3, PRF1, and GZMB mRNAs and decreasing levels of NCAM1 mRNA toward term pregnancy. These results indicate that NK cell markers are expressed in the uterine endometrium during the estrous cycle and pregnancy in a stage- and pregnancy status-dependent manner. NK cells may play an important role in the establishment and maintenance of pregnancy by regulating the endometrial immune environment in pigs. Further analysis of localization and function of NK cells at the maternal-conceptus interface is needed.

S100 protein family is small calcium-binding proteins with EF-hand motif and comprises more than 20 proteins in human. S100 proteins are involved in intracellular and/ or extracellular signaling, such as proliferation, differentiation, apoptosis, calcium homeostasis, inflammation and migration. Previously, we have shown that the expression of S100A15A mRNA in the endometrium is increased on Day 12 of pregnancy compare to the estrous cycle. However, the expression and regulation of S100A15A have not been determined in the uterine endometrium during the estrous cycle and pregnancy in pigs. Therefore, we analyzed the expression and regulation of S100A15A mRNA in the uterine endometrium during the estrous cycle and pregnancy. Real-time RT-PCR analysis showed that S100A15A mRNA was expressed in the uterine endometrium during the estrous cycle and pregnancy with higher levels on Day 12 of pregnancy than the estrous cycle. To investigate the effects of steroid hormones and interleukin 1 beta (IL1B) on the expression of S100A15A mRNA, endometrial tissue explants from Day 12 of the estrous cycle were treated with steroid hormones, estradiol and progesterone, or IL1B. The levels of S100A15A mRNA were increased by estradiol and IL1B. These results showed that S100A15A was expressed in the uterine endometrium in a pregnancy stage-specific manner and the expression of S100A15A was regulated by estradiol and IL1B of conceptus origin, suggesting that S100A15A may play a key role in the establishment of pregnancy in pigs.

For successful pregnancy, the appropriate interactions between the maternal endometrium and the implanting conceptus are essential during the implantation period. In pigs, the implanting conceptus produces estrogens, interleukin 1 beta (IL1B), interferon delta (IFND) and interferon gamma (IFNG) during early pregnancy. Estrogens are produced by CYP19A1, a key enzyme for estrogen biosynthesis, and play a role in maternal recognition of pregnancy in pigs. IL1B is known to be involved in production and transport of prostaglandins in the endometrium. The role of IFND and IFNG during the implantation period is not fully understood. Also, localization of CYP19A1, IL1B, IFND and IFNG in the implanting conceptus is not fully understood. Thus, this study determined the localization of these molecules in conceptus during the peri-implantation period in pigs. We performed in situ hybridization and immunohistochemistry using conceptus on Days 12 and 15 of pregnancy. CYP19A1 were detected mainly in trophectoderm of conceptus on Days 12 and 15 of pregnancy. IL1B was expressed in trophectoderm of Day 12, but not Days 15. IFND mRNA and protein were localized to trophectoderm and endoderm of Days 12 and 15 with stronger immunostaining signal on trophectoderm than endoderm. IFNG mRNA and protein were mainly localized to trophectoderm, but not endoderm, on Days 12 and 15 of pregnancy. These results showed that CYP19A1, IL1B, IFND and IFNG were localized to conceptus, suggesting that these molecules secreted from conceptus during the peri-implantation period are dynamically regulated during conceptus development in pigs. Further study is needed to analyze the function of these molecules in the uterine endometrium during early pregnancy.

The objective of this study was to investigate the effects of alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. ALA was diluted using ethanol and cumulus- oocyte complexs (COCs) were incubated in IVM medium containing 50 μM of ALA or 0.9% ethyl alcohol for 44 h. After in vitro maturation, denuded oocytes were stained using aceto-orcein solution for evaluation of nucleic maturation. Then, COCs were maturated using IVM medium with 50 μM ALA and fertilized with spermatozoa. Fertilized embryos were cultured in IVC medium with 25 μM ALA at different stage of embryogenesis: T1, without ALA both early- and late-embryogenesis; T2, with ALA at late embryogenesis; T3, with ALA at early embryogenesis; T4, with ALA for both early-and late-embryogenesis. Embryo development was evaluated by cleavage rates, blastocyst formation and total cell number. In results, nucleic maturated oocytes were significantly increased by ALA treatment groups compared with control groups (p<0.05). The treatment groups of T2 and T3 were significantly increased cleavage rate than T4 group (p<0.05), whereas there were no significant difference compared to control group and blastocyst formation was not influenced by ALA during IVC. Interestingly, total cell number of blastocyst in T3 group was significantly higher than in T3 treatment group (p<0.05). This present study show that treatment of ALA during IVM and IVC enhanced the oocyte maturation and embryo quality, especially, embryo development is positively influenced by ALA during early-embryogenesis. However, long-therm treatment of ALA negatively affect to porcine embryo development.

In vitro production of pig embryos has been emerged for xenotransplantation due to physiological properties. Sirtuins play a crucial role in several cellular processes including DNA repair, apoptosis, cell cycle, and determining lifespan as NAD+ dependent class III histone deacetylase. Previous studies have shown that sirtuin inhibition induces embryonic developmental arrest and oxidative stress in porcine and murine models. Also, sirtuins regulate autophagy pathways through the deacetylation of ATG proteins and transcriptional factors. However, sirtuins-mediated mechanisms have not been examined in blastocysts. Therefore, we investigated the relationship between sirtuin inhibition and autophagy in porcine model. Embryos were cultured in 100 μM sirtinol (SIRT1/2 target inhibitor)-treated NCSU-23 media after in vitro fertilization. Treatment with sirtinol significantly decreased the rates of morula (21.34±1.84 vs. 11.89±2.01%), blastocyst formation (17.18±1.81 vs. 9.00±2.02%), and total cell number (50.80±1.47 vs. 37.71±1.79). Especially, expanded blastocysts (9.90±1.56 vs. 2.92±0.94%) were barely observed in sirtinol treated group. The level of Sirt2 expression was significantly decreased in sirtinol-treated group compared to control. Also, sirtinol induced autophagy by increasing LC3 transcript and LC3 protein in treatment group. Beclin1 and ATG5 expressions showed a slight increase in sirtinol-treated group. Finally, sirtuin inhibition by sirtinol showed significantly increased TUNEL indices (6.55±0.84 vs. 11.44±0.81) and fragmentation indices (0.33±0.05 vs. 1.40±0.30). Similarly, the level of BCL2L1 was lower, while the level of Caspase-3 expression was significantly elevated in the treated group compared to controls. Therefore, these results suggest that sirtuin inhibition may play an important role in porcine preimplantation development and embryo quality through regulation of autophagy and apoptosis pathways.

Poly(ADP-ribosyl)ation (PARylation) is related to DNA repair, chromatin modification, and apoptosis and catalyzed by poly (ADP-ribose) polymerases (PARPs). Recent studies have shown that PARylation affects preimplantation development and autophagy mechanism in pig embryos. However, the role of PARylation during in vitro maturation (IVM) has not yet been investigated. To study the effect of PARylation, cumulus-oocyte complexes were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during IVM. Nuclear maturation of oocytes were showed no significant difference in all stages (GV, MI, MII), but full expansion of cumulus cell was significantly decreased (11.05±1.09 vs. 48.40±0.67%) in 3-ABA treatment group. To evaluate mRNA levels of maturation and expansion related genes, we analyzed 3-ABA treated oocytes and cumulus cells. GDF- 9, BMP15, COX-2, PTX3 expression were upregulated but levels of HAS2, TNFAIP6 were downregulated in treatment group. To evaluate the effect of PARP inhibitior on embryo development and blastocyst quality, we experimented parthenogenetic activation (PA). As a results, the developmental rate of PA embryos was significantly decreased in 3-ABA group. Furthermore, TUNEL index (4.80±0.66 vs. 2.42±0.35) and total apoptotic index (6.44±0.81 vs. 3.08±0.51) was significantly increased, while Total cell number was decreased (41.12±2.10 vs. 50.38±2.27). Therefore, these results suggested that PARylation plays an important role in IVM of porcine oocytes.

일반적으로 Isoproterenol은 melatonin의 합성과정에 있어서 melatonin을 활성화 시켜 그 활성도를 향상시키는 것으로 알려져 있다. Melatonin은 포유동물의 송과선에서 분비되 는 호르몬으로서 특히 난포액에서 높은 농도로 검출되며, 난자의 체외배양의 경우, 강력 한 활성산소가 청소부 작용을 하여 난자의 체외발달율을 개선시키는 역할을 한다. 따라 서 본 연구에서는 isoproterenol과 melatonin의 적정농도 확인과 난포란의 quality가 낮은 3등급 난자의 체외발달율 개선효과 여부를 검토하고자 melatonin 10—7∼11—11 및 isoproterenol 10—10∼10—14몰 농도로 체외성숙 배양액에 첨가하고 난포란의 등급을 구분하여 단위발생을 유기하였다. 뿐만 아니라 체외 배양액에 첨가하여 등급이 낮은 돼지 난포란 유래 단위발생란의 분할율 및 배반포기 발달율을 조사하였다. 도축장으로부터 채집한 돼지 난소의 난포에서 난포란을 채취하여 난구세포층이 3∼5층 이상 충실하게 부착되어 있는 난포란을 Grade І(1∼2등급), 난구세포층이 1∼2층 이하 및 부분적으로 나화되어 있는 난포란을 Grade III(3∼4등급)으로 등급을 구분하여 전기자극법 으로 자극을 가한 다음, 2 mM의 6-DMAP 용액으로 2시간 동안 처리하여 단위발생을 유 도하였다. Isoproterenol(—10, —12, —14몰)과 melatonin(—7, —9, —11몰)을 체외성숙 및 체 외 배양시에 각각 병용첨가하였다. 체외성숙 배양액은 TCM-199 배양액을 기본배양액으 로 하여 각종 호르몬을 첨가하여 사용하였으며, 체외배양액은 PZM-5에 3 mg FAF-BSA 를 첨가하여 38.5℃, CO2 배양기에서 7일간 배양을 실시하였다. 체외 성숙 및 배양시에 melatonin 10—10몰 및 isoproterenol 10—12몰을 첨가하였을 때, 3등급 난자의 체외 성숙율 이 79.3%로서 1등급(82.6%) 난포란과 차이가 없을 정도로 개선되었으며, 배반포기로의 체외발달율은 melatonin 10—10몰 및 isoproterenol 10—12몰이 67.0%(1등급) 및 50.2%(3등 급)로서 1등급이 유의적(p<0.05)으로 높은 발달율을 보였으나, 다른 처리구에서는 1, 3등 급 간에 차이가 없었지만 개선되는 경향을 보였다. 이상의 결과로 볼 때, melatonin과 isoproterenol의 첨가농도는 melatonin 10—10몰 및 isoproterenol 10—12이 적정한 농도인 것으로 판단되며, 1, 3등급 간에 차이가 없음을 확인 하였다. 따라서 이들을 첨가하면 난포란의 활용도 제고에 크게 기여할 수 있을 것으로 생각된다. Key words)

Heterozygous status of recipient mitochondria and donor mitochondria in somatic cell nuclear transfer (SCNT) embryo is a quite defective. To solve the problem, our previous research has proposed that treatment of 2’,3’-dideoxycytidine (ddC) for in vitro maturation of oocyte induces the depletion of mtDNA in bovine oocytes. Bovine oocytes were matured in 20 μM ddC for 24h at 39℃ in a humidified atmosphere of 5% CO2. The second meiotic metaphase (MⅡ) oocytes were selected for oocyte activation and then activated by a combination of 5 μM inomycin for 5 min and 2 mM 6-DMAP for 3 h. GFP-tagged exogenous mitochondria were injected into the parthenogentic embryos using a 15 μM of injection pipette at 16 h after oocyte activation. Embryos were cultured in mSOFaa medium and changed medium every 2 days. At 7 days, the development rate of embyos was measured. GFP and hoechest were observed by a fluorescence microscope. As a result, the maturation rate of bovine oocytes were not significantly lower than that in the control oocyte. In addition, the development of embryos treated with 20 mM ddC for in vitro maturation and injected exogenous mitochondria was not different from that of the control. However, expression of GFP was showed only in the embryos injected exogenous mitochondria. In conclusion, we transmitted exogenous mitochondria into mtDNA depleted bovine oocytes.

A time-lapse monitoring system has predictive value for selecting good-quality embryos with the highest implantation potential. Using this new tool, we investigated the developmental potential and developmental kinetics of bovine parthenogenetic (PA) and two types of somatic cell nuclear transfer (NT) embryos. Bovine non-transgenic ear cells (bECs) or transgenic cells (bTGCs) were used as donor cells. The cleavage and blastocyst development rates did not significantly differ among the PA, NT-bEC, and NT-bTGC groups, and first cleavage occurred an average of 19.3 h (n=70), 21.6 h (n=60), and 21.3 h (n=62) after activation, respectively (20.4 h (n=192) for all embryos). When embryos were classified into early cleaving (≤ 20 h) and late cleaving (> 20 h) groups, the blastocyst formation rate was much higher in the early cleaving groups (PA, 46%; NT-bEC, 50%; NT-bTGC, 39%) than in the late cleaving groups (PA, 18%; NT-bEC, 23%; NT-bTGC, 28%), while the percentage of embryos whose development was blocked between the 2- and 8-cell stages was increased in the late cleaving groups. The percentage of embryos classified as early cleaving with a normal morphology was 2-fold higher in the PA group (20.0%, n=14) than in the NT-bTGC group (9.7%, n=6). The timing of each developmental stage varied widely; the timing of first cleavage varied from 7.6 h in the PA group to 34.5 h in the NT-bEC group and the timing of expanded/hatching blastocyst appearance varied from 141.6 h in the PA group to 196.3 h in the NT-bTGC group, differences of 26.9 and 54.7 h, respectively (PA>NT-bEC>NT-bTGC). These results demonstrate that time-lapse monitoring provides novel data regarding individual embryo developmental kinetics and helps to predict developmental potential for improved bovine NT embryo selection based on early cleavage (< 20 h) and normal morphology.

One of the reasons about low quality of in vitro matured (IVM) oocytes than those of in vivo is the oxidative stress caused by external oxygen and incomplete antioxidant system. This study was performed to examine the effects of β-cryptoxanthin (BCX, anti- oxidative reagent) on porcine oocyte during IVM and further in vitro developmental potential. The oocytes were matured in IVM medium containing 0, 0.1, 1, 10 and 100 μM BCX (control, 0.1, 1, 10 and 100 B, respectively). The rate of oocyte maturation was higher in 1 B than in control (p<0.1), while that of other BCX treated groups were similar to control. After IVM, the cytoplasmic reactive oxygen species (ROS) expression level in 1 B was the lowest among all groups (p<0.05), while other BCX treated groups were similar to or higher than control. Also, at the classified oocyte maturation stages (GVBD, MⅠ and MⅡ), 1 B significantly decreased ROS and increased GSH level than control (p<0.05). In addition, the relative mRNA expression level of antioxidant genes, SOD1 and PRDX5, were higher in 1 B than in control (p<0.05). As the provitamin A, the relative mRNA expression level of retinoic acid receptor genes, ITGB7 and RXRA, were significantly increased in 1 B than in control (p<0.05). After parthenogenetic activation, the cleavage rates were not different between control and 1 B, however, the blastocyst formation rate was higher in 1 B than in control (p<0.01). In embryo quality, while the DNA fragmentation of blastocysts was similar between control and 1 B, the total cell number (p<0.1) and the relative mRNA expression level of pluripotency marker genes, Pou5f1 and CDX2 (p<0.05), and anti-apoptosis genes, Bcl2L1, Bcl-xl and BIRC5 (p<0.05), were significantly increased in 1 B than in control. These results demonstrate that BCX is helpful for decreasing oxidative stress in porcine oocytes and improves their quality and developmental potential.

Sperm treatment methods affect sperm fertility and in vitro developmental potential of in vitro fertilized (IVF) embryo. Jeju Black Cattle (JBC) is an indigenous species of Korea and IVF system is very important for mass production and industrialization of JBC. In order to find out the most suitable sperm treatment method, the following methods of percoll gradient, triladyl and percoll-triladyl combination (P, T and PT, respectively) were used to sperm treatment. At 2 days after IVF, the fertilization rate was higher in T and in PT groups than in P group. The blastocyst formation rate was significantly increased in T group than P and PT groups. The total cell number was higher in T and PT groups than in P group. The blastocyst formation rate was increased in T group. When we performed IVF after IVM treatment of individual ovaries, in comparison of the development rates of donor and non-donor cows, there was no significant difference. According to grade (1++, 1+, 1, 2), the development rate was the highest in 1++ grade cattle, followed by 1, 1+ and 2 grade. Also, in vitro developmental potential by carcass weight was the highest at the 300～350 kg, followed by 300 kg or less, 350～400 kg, then 400 kg more over. As a result, the most effective sperm processing was resulted in PT method, and the development of IVF embryos was more effective when the carcass grade was high and the carcass weight was about 350 kg. This study demonstrates that JBC sperm can be effectively recovered with simple and efficient PT method and from this protocol, high quality embryos can be produced in vitro.

Allicin (AL) is one of the chemical substance from garlic and has strong antioxidant activity and considered to represent anti-aging effect in vitro. The objective of this study was to investigate the effects of allicin treatment during porcine oocyte aging and their in vitro developmental competency after somatic cell nuclear transfer (SCNT). Porcine oocytes were maturated in vitro for 44 h (control), 44+24 h and 44+24 h with 0, 0.1, 1, 10 and 100 μM allicin (0, 0.1, 1, 10 and 100 AL, respectively). We investigated the effects of appropriate concentration of allicin on nuclear, cytoplasmic maturation and the developmental capacity of aged porcine oocytes. The 1 AL significantly increased maturation rate and normal spindle formation rate compared to control and 0 AL. The mRNA expression of cytoplasm maturation marker genes (MOS, BMP15, GDF9 and CCNB1) in 1 AL was higher than control and 0 AL. Allicin enhanced the rearrangement of abnormal spindle in aged oocytes. Parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryos development in 1 AL were significantly increased compared to others. Allicin treatment on aged oocytes improved their poor developmental capacity by rearranging the cytoskeleton. Thus, the allicin is effective agent to prevent the poor developmental competence of porcine aged oocytes and enhanced PA and SCNT embryo developmental capacity. Therefore, AL may be helpful for improve the aged oocytes quality to use on in vitro experiments.