소에서 설사 등의 소화기질환, 폐렴 등의 호흡기질환, 유산 등의 번식장애를 유발하는 소 바이러스성 설사병(Bovine Viral Diarrhea, BVD)은 소 사육농가에서 경제적인 손실을 입히는 대표적인 생산성 저하 질병 중의 하나인데, 특히 유산, 사산 및 수태율 저하를 유 발하여 번식우 사육농가에 직접적인 피해를 입히고 있는 실정이다. 농림축산검역본부에 서는 BVD로 인해 1마리당 50,000원 정도의 손실로 연간 손실액이 최소 1천억 이상이라 고 추정하였고, 미국은 급성 BVD 감염으로 소 1마리당 약 400달러의 피해가 발생한다고 할 만큼 소 사육농가에 막대한 피해를 입히고 있지만, 우리나라에서 아직 법정 가축전염 병으로 지정되지 않아 체계적인 발생 상황이 확인되지 않고 있는 실정이다. 본 연구에서 는 한우 및 젖소 사육 농가를 대상으로 소 바이러스성 설사의 항원 양성률을 분석하여 소 바이러스성 설사병의 발생 현황을 확인하고자 하였다. 본 실험을 위해 4개도(강원, 전 북, 경남, 제주) 9개 목장에서 사육하고 있는 한우 송아지 및 젖소 육성우를 대상으로 분 변을 이용하여 PCR 방법으로 농장별, 생후 일령별 항원 양성률을 조사하였다. 한우 송아 지의 BVD 바이러스 항원 양성률을 조사한 결과 전체 조사 두수의 60.0%가 양성을 나타 내었고, 농장별로는 10.0%∼81.8%를 나타내어 농장별로 양성률에 많은 차이를 나타내었 다. 젖소 육성우의 경우 28.7% 양성률을 나타내었는데, 농가별로는 0%∼65.0%로 한우 송 아지와 마찬가지로 농장별로 차이를 보이고 있었다. 한우 송아지의 생후 일령(주령)별 양 성률을 조사한 결과 조사된 한우 송아지의 54.8%가 항원 양성률을 나타내었는데, 1주령 80.0%, 2주령 33.3%, 3∼4주령 45.5%, 5∼8주령 60.0%, 9주령 이상 50.0%를 나타내어 송 아지의 전체 연령대에 다양하게 항원 양성반응을 나타내었다. 소에서 번식장애 등의 손실 을 입히고 있는 BVD는 우리나라에서 예방백신이 개발되어 활용되고 있지만 소 사육농가 에서는 구제역 등과 같은 전염병에 비해 현실적으로 피해를 실감하지 못하고 있는 질병 이기도 하다. 따라서 본 연구 결과를 바탕으로 농가에서 임신 예정우, 송아지를 대상으로 예방백신 접종을 권장함으로써 그 피해를 경감시킬 수 있는 질병으로 사료된다.

말은 단위 동물을 가진 초식동물로 반추 동물과는 달리 한 번에 다량의 농후사료를 섭취할 경우 질환 증상이 나타날 수 있는데 산통이 대표적인 증상으로 전체 말의 약 4% 에서 발생하며 폐사율이 28%에 이르는 것으로 보고되고 있다(Mary 등, 1997). 말의 안정 적인 번식을 위해서는 에너지 함량이 높은 농후사료 공급 비율을 증가시킨 사양 프로그 램의 개발이 필요하므로 본 연구는 번식마의 소화 생리를 고려한 사료 급여 방법 구명하 기 위하여 수행되었다. 공시축은 제주산마 14두를 공시하여 체중의 1.5%, 2%, 2.5%의 농 후사료를 일일 2회에 나누어 급여하였고 건초는 자유채식으로 8주간 급여하여 그에 따른 체중 변화와 분 pH, D-dimer, 피브리노겐 등을 조사하였다. 총 증체량은 1.5%, 2%, 2.5% 에서 각각 21.6±13.4kg, 28±20.6kg, 42.6±15.2kg였고, 일일 증체량은 각각 0.4±0.25kg, 0.52±0.38kg, 0.8±0.28kg이었다. 분 pH는 시험 개시 후 8주째에 1.5%, 2%, 2.5% 급여구에 서 각각 5.93±0.29, 5.94±0.18 및 5.87±0.21로 전반적으로 낮은 수치를 나타내었다. 혈액 내 피브리노겐 수치는 1.5%, 2%, 2.5% 급여구에서 각각 238±20.2g/dl, 222±26.8g/dl 및 222±20.7g/dl로 정상 수준을 보였다. D-dimer 수치는 1.5%, 2% 급여구는 모두 정상수치 를 나타냈으나 2.5% 급여구에서는 60%에서 정상보다 높은 수치를 나타났다. 시험 개시 후 8주까지 모든 처리구에서 임상적으로 산통이 발생하지 않았으며 임상적으로 산통이 나타나진 않았으나 2.5% 급여구에서 D-dimer 수치가 높은 수치를 나타내는 경향이 있었다.

말은 비반추 초식동물로서 위 용적이 작아 많은 양의 농후사료를 섭취할 경우 산통 증 상이 나타나는 것으로 알려져 있다. 산통은 말 소화기관의 특이한 구조로 인하여 복부의 통증을 나타내는 증상으로 농후사료 과다급여 혹은 모래 섭취 등 다양한 이유로 발생한 다. 산통의 발생 빈도는 전체 말의 약 4~10%이며 이중 6~28%의 폐사하는 것으로 알려 져 있다. 말의 안정적인 번식을 위해서는 농후사료 적정 수준으로 증가시킨 사양 프로그 램의 개발이 필요하므로 본 연구에서는 번식마의 소화 생리를 고려한 사료 급여 방법 구 명하기 위하여 수행되었다. 공시축은 제주산마 13두를 공시하여 체중의 2.5%의 농후사 료를 일일 2회, 3회, 4회에 나누어 급여하였고 건초는 자유채식으로 12주간 급여하여 그 에 따른 체중 변화와 분 pH, D-dimer, 피브리노겐 등을 조사하였다. 총 증체량은 2회구, 3회구, 4회구에서 각각 55.8±17.2kg, 54.4±17.5kg, 63±9.5kg였고 일일증체량은 각각 0.69±0.21kg, 0.67±0.21kg, 0.78±0.11kg이였다. 분 pH는 2회구, 3회구 및 4회구에서 각각 6.13±0.06 , 6.18±0.07 및 6.15±0.32로 정상 수준이였고, 혈액 내 피브리노겐 수치는 2회구, 3회구 및 4회구에서 각각 194.6±23.4, 197±37.4 및 191±22.7g/dl로 정상 수준을 보였다. D-dimer를 시험 개시 후 12주째에서 측정한 결과 정상보다 높은 수치를 나타낸 시험축 이 2회구에서는 60%, 3회구에서는 60%였고, 4회구에서는 33%가 나타났다. 농후사료 2.5%를 급여한 모든 처리구에서 임상적으로 산통이 발생하지 않았으며 분 pH, 피브리노 겐은 정상범위에 있었으나 급여횟수가 적은 공시축에서 D-dimer가 정상수치보다 높은 경향을 나타내었다.

소의 수정란 생산과 이식에 대한 연구는 한우의 개량과 증식 그리고 한우 유전자원의 안정적인 관리로 산업적 활용성을 증대 시킬 수 있다. 따라서 본 연구에서는 한우의 생체 에서 난포란을 채란에 따른 효율성을 높이기 위해서 연구를 수행하였다. 생체난포란 채란 을 위한 한우 공란우는 한우연구소에서 사육중인 한우에서 실시하였다. 생체 내 난포란의 관찰은 MyLabTM30VETGOLD(Esaote, Genova, ITALY) 및 탐촉자(EC123; Micro-Convex 9∼3 MHz, Esaote, Genova, ITALY)는 6.6 MHz convex scanner를 사용하였고, 난포란 채란에 사용된 주사침은 19G 주사침을 사용하였다. Monitor Image에 고정된 2 mm 이상 의 난포에서 난포의 수량을 확인하고 난포란을 흡인하였다. 흡입된 난포란은 2∼3회 washing으로 혈액 등의 이물질을 제거하여 실체현미경 하에서 회수하였고, 난포란의 등 급분류는 세포질 균일도와 난구세포의 부착 정도에 따라 평가기준을 1등급에서 3등급까 지 분류하였다. 회수된 난포란의 체외성숙배양은 TCM 199 기본배양액에 소 태아혈청(Fetal Bovine Serum) 0.5%와 LH, FSH, FGF, EGF 첨가하여 22시간 동안 배양하였고, 체외수정은 IVF 100(IFP, Japan) 배양액 50μl 미소적에 성숙 난포란 20개씩 넣어서 체외수정을 실시 하였다. 체외수정을 위해서 KPN 동결정액을 사용하였고 수정을 위한 정자의 최종 농도 는 2x106/ml로 6시간 동안 체외수정을 유도하였다. 체외배양은 조건은 5% O2, 5% CO2, 그리고 90% N2 와 38.5°C 인큐베이터에서 배양하였다. 수정율은 체외수정 후 48시간째 확인하였고 배반포 수정란 발달율은 체외수정 후 192시간째 확인하였다. 농후사료 급여량 에 따른 난포란의 회수효율은 2.0kg/일 급여시 초음파상에서 채취가능 난포란은 9.6개, 3.0kg일 때는 8.8개로서 유의적인 차이는 없었으나, 2.0kg 급여시에 다소 높은 난포란을 확인하였다. 생체 채취된 난포란의 체외배양에 따른 수정란의 발달율은 IVD 배양액에서 15.6%, CR1aa 26.2%, SOF 37.1% 발달율로서 IVD 배양액보다 SOF 배양액에서 유의적 으로(P<0.05) 높은 발달율을 보였다. 배양액 종류에 따른 동결 융해후의 생존율은 66.7%, 64.7%, 70% 각각 나타내었다. 생체난포란을 활용한 수정란이식을 위해서 체외배양시스템 의 안정적인 시스템 구축이 필요하며 생체채취 난포란의 회수를 위한 시술자의 기술적인 숙련도가 상승되었을 때 효율성을 제고할 수 있을 것으로 시료된다.

소에서 저수태 발생율은 5∼35%로 지역, 조사방법 및 연구자 등에 따라 다양하게 보고 되고 있다(Purohit, 2008). 저수태의 원인으로서는 비정상적인 번식기관, 자궁 내 감염, 호 르몬 이상, 사양관리 부적합, 복합적 요인 등이 관여하고 있는데(Singh 등, 2008), 이 중에 서도 자궁 내 감염의 요인이 2.4∼20.0%로 비교적 높게 보고되고 있다(Rao, 1981). 자궁질 환을 가진 대부분의 암소들은 황체단계가 연장됨에 따라 무발정형태로 나타나게 되므로 적극적인 대처가 요구된다는 점에서, 호르몬 투여방법을 포함하여 PGF2α나 estradiol의 투 여 그리고 항생물질이나 povidone-iodine(PVP-I)의 자궁 내 주입과 같은 많은 치료방법이 적용되고 있다. 그러나 호르몬 잔류, 항생제 내성균 및 처리 중단시기 등의 문제점들을 가 지고 있다. PVP-I는 처리 중단시기가 필요하지 않고, 과도한 투여의 경우를 제외하고는 우유로 전이되지 않으며, 구입이 쉽고, 저렴하며, 투여하기가 용이하다는 실용성을 가지고 있기 때문에 매우 고무적인 방법이라고 할 수 있다(Rayos 등, 2002). 또한 자궁내막의 환 경을 개선하고, 암소의 자궁수촉을 촉진시키기 때문에 PGF2α를 생산하는 역할이 빨리 회 복된다(Ahmed와 Elsheikh, 2014). 그러나 PVP-I는 농도가 중요한데, 농도가 너무 높으면 자궁벽을 자극할 수 있고 문제를 더 키울 수 있는 반면에, 농도가 너무 약하면 효과가 나 타나지 않거나, 어떤 유형의 미생물이 제거되는 대신에 다른 기회적 미생물이 점유하기에 적합한 환경을 제공할 수가 있다(Rayos 등, 2002). 자궁내막염을 가진 암소의 경우에는 자 궁 내에 존재하는 화농과 유기물의 잔재가 PVP-I 용액의 살균효과를 저해할 수 있다는 부정적인 견해도 있으나(Nakao 등, 1988), 대부분의 연구자들은 1% 및 2% PVP-I 용액이 번식효율을 향상시키는데 매우 효과적이라고 보고하였다(Mido 등, 2016; Ahmed와 Nour, 2015; Ahmed 등, 2013; Knutti 등, 2000; Edwell 등, 2000; Rayos 등, 2002; Koujanz 등, 1996). 본 연구에서는 2% PVP-I 용액을 저수태우의 자궁 내에 주입하여, 발정발현 및 수 태율에 미치는 영향을 구명하고자 수행하였다. 저수태우에 2% PVP-I 용액을 50mL 자궁 내에 주입하여 자연발정 비율, 유기발정(PGF2α)에 의한 발정발현율, 자연 및 유기발정에 의한 수태율, 황체중기 및 황체중기 이외의 단계에 따른 자연발정 발현율을 조사하였다. PVP-I 처리 후 자연발정 비율은 27.3%(3/11두)였고, 유기발정에 의한 발정발현율은 87.5%(7/8두)였으며, 자연 및 유기발정에 의한 수태율은 63.6%(7/11두)였다. 황체중기(발정 주기 5∼10일) PVP-I 처리에 따른 자연발정 발현율은 60%(3/5두)였고, 황체중기 이외에 PVP-I 용액을 처리한 경우의 자연발정 발현율은 0%(0/6두)였다.

High reproductive efficiency is a prerequisite for dairy animals in order to optimize dairy production. An accurate and early pregnancy diagnosis is a crucial aspect for better reproductive management in livestock. The indigenous, visual and clinical methods have various limitations including accuracy, sensitivity, specificity, later stages of applicability and requires highly skilled manpower. This hurdles the researchers to ensue further research on developing novel early pregnancy diagnostics for dairy animals. However, the advantage of molecular techniques like proteomics has given a new hope to look for pregnancy biomarkers in animal research. In this study, we assayed the pregnancy-associated glycoprotein (PAG) levels using anti-bPAG antibody. Serum plasma samples were collected 30, 60, 90 and 120 days after insemination. Cattle diagnosed pregnant with singleton are used for this experiment in which blood samples were collected. The plasma PAG level is gradually increased after insemination to until 60 days and drastically decreased after that. The PAG level was approximately 2-fold greater during 30 days compared with 60 days. These results concluded to validate our other pregnancy diagnosis methods including circulating microRNA and cell free DNA concentration (data not shown). Despite this proteomic approach, validating all other molecular technique results may give the exact time for early pregnancy diagnosis.

Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.

Progesterone regulates endometrial functions to support implantation, placentation, and fetal/placental development in the uterus. It is known that actions of progesterone are mediated by nuclear progesterone receptor (PGR), using the signaling pathway referred to the genomic pathway. However, all physiological progesterone actions cannot be explained by the genomic pathway via PGR, and it is understood that there are non-genomic actions of progesterone though membrane progesterone receptors, progesterone receptor membrane components (PGRMCs) and progestin and adipoQ receptors (PAQRs). The expression and localization of PGRMCs and PAQRs has been reported in female reproductive tissues of several species such as human, mouse and cattle. Previously, we have shown that PGRMCs and PAQRs are expressed in the porcine uterine endometrium during the estrous cycle and pregnancy. However, the regulatory mechanism for expression of PGRMCs and PAQRs in the uterine endometrium has not been studied in pigs. Thus, to understand the regulatory mechanism of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 expression in the uterine endometrium during the estrous cycle and pregnancy in pigs, we determined the effect of steroid hormones estrogen and progesterone on expression of PGRMCs and PAQRs using the endometrial tissue explants for immature pigs. Levels of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 mRNAs were increased by increasing doses of progesterone, but not by estradiol in the uterine endometrium. Blocking PGR by treatment of RU486, a progesterone receptor antagonist, increased levels of endometrial PGRMCs and PAQRs mRNA. These data showed that membrane progesterone receptors were induced by progesterone in the uterine endometrium, suggesting that these membrane progesterone receptors may play an important role in mediating progesterone actions in the uterine endometrium for regulation of the estrous cyclicity and pregnancy. [Supported by the Next Generation Biogreen 21 Program (# PJ01119103), Rural Development Administration, Republic of Korea]

For the establishment and maintenance of successful pregnancy the maternal immune system must tolerate semi-allogenic fetus during pregnancy. Several mechanisms explaining immune tolerance have been proposed. Among those, it has been suggested that the CD40/CD40L system is involved in immune tolerance in several tissues. However, expression and function of CD40/CD40L in the maternal-fetal interface during pregnancy have not been studied in pigs. Thus, this study determined expression and localization of CD40 and CD40L in the uterine endometrium during pregnancy in pigs. We obtained uterine endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR analysis showed that levels of CD40L mRNA expression during pregnancy increased on D15 of pregnancy and decreased thereafter whereas levels of CD40 mRNA was highest on D30 of pregnancy. Localization of CD40 and CD40L proteins by immunohistochemistry showed that CD40 was localized to vascular endothelial cells with strongest signal intensity on D15 of pregnancy, and CD40L was localized to luminal epithelial cells on D15 of pregnancy and amniotic membrane during mid- to late pregnancy. To determine the effect of IFNG on CD40 and CD40L expression, we took advantage of endometrial explant culture using tissues from D12 of the estrous cycle, and found that CD40 was up-regulated by IFNG in a dose-dependent manner. These results showed that CD40 and CD40L were expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and IFNG induced CD40, indicating that the CD40/CD40L system may be important for establishment and maintenance of pregnancy in pigs. [Supported by the Next Generation BioGreen21 Program (#PJ01110301), Rural Development Administration]

S100As are calcium-binding proteins with two EF-hand calcium-binding motifs. In several studies, S100A proteins are described to play important roles in pro-inflammatory responses including damage-associated molecular pattern (DAMP) signaling and in the establishment of pregnancy. However, the role of S100As have not been determined in the uterine endometrium during the estrous cycle in pigs. Thus, this study was performed to investigate expression and regulation of S100A8, S100A9, and S100A12 in the uterine endometrial tissues during the estrous cycle in pigs. Real-time RT-PCR analysis showed that S100A8, S100A9, and S100A12 mRNAs were expressed in the uterine endometrium during the estrous cycle with higher levels on days 15 and 18 of the estrous cycle than other days of cycle. To investigate the effects of steroid hormones, estradiol (E2) and progesterone (P4), on expression of S100A8, S100A9, and S100A12 mRNAs, endometrial tissue explants from immature pigs were treated with steroid hormones. Levels of S100A8, S100A9, and S100A12 were increased by the treatment of P4, and the increased levels of S100A8, S100A9, and S100A12 by P4 were not inhibited by the treatment of progesterone receptor antagonist, RU486. However, levels of S100A8, S100A9, and S100A12 were decreased by treatment of MEK inhibitor, U0126. These results exhibited that S100As were expressed in the uterine endometrium during the estrous cycle in a cyclic stage-specific manner, and their expression was affected by P4. These suggest that S100As may play an important role in endometrial function during the proestrous period of the estrous cycle in pigs. [Supported by the Next Generation Biogreen 21 program (#PJ01119103), Rural Development Administration, and by Korea Research Foundation (#2015R1D1A1A01058356)]

The migration, adhesion, and proliferation of conceptuses during pregnancy are tightly controlled processes that are mediated by various factors including cytokines, growth factors, and hormones. Among many factors, chemokines play key roles in lymphocyte trafficking, cellular proliferation, vascularization, and embryogenesis in many mammalian species. Especially, it has been shown that C-X-C chemokine ligand 12 (CXCL12) plays an important role in early pregnancy by promoting trophoblast invasion, proliferation, and differentiation through its receptor, C-X-C chemokine receptor 4 (CXCR4) in humans. However, expression and function of CXCL12 in the uterine endometrium during pregnancy have not been well studied in pigs. Thus, we determined expression of CXCL12 and its receptor, CXCR4, in the uterine endometrium during the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on day (D) 12 and D15 of the estrous cycle and D12, D15, D30, D60, D90, and D114 of pregnancy, conceptus tissues from D12 and D15 of pregnancy, and chorioallantoic tissues from D30, D60, D90, and D114 of pregnancy. Real-time RT-PCR analysis showed that levels of CXCL12 and CXCR4 mRNAs changed in the uterine endometrium during pregnancy. Levels of CXCL12 and CXCR4 mRNAs on D15 of pregnancy were higher than those on D15 of the estrous cycle. After D15 of pregnancy levels of CXCL12 and CXCR4 mRNAs gradually decreased toward term of pregnancy, and CXCL12 and CXCR4 were expressed in the chorioallantoic tissues during the mid- to late pregnancy. CXCL12 and CXCR4 mRNAs were expressed in chorioallantoic tissues during mid- to late pregnancy, and RT-PCR analysis showed that CXCL12 and CXCR4 mRNAs were detectable in conceptus on D12 and D15 of pregnancy. Immunohistochemistry showed that CXCL12 proteins were localized to endometrial luminal and glandular epithelial cells during the estrous cycle and pregnancy, and to chorionic epithelial cells during mid- to late pregnancy. Abundance of CXCL12 mRNAs, but not CXCR4, in the uterine endometrium was increased by the treatment of IFNG. These results showed that CXCL12 and CXCR4 were expressed in the uterine endometrium, conceptus, and chorioallantoic tissues and IFNG increased endometrial CXCL12 expression in pigs, suggesting that CXCL12 and its receptor may play a key role in regulation of the establishment and maintenance of pregnancy by affecting the conceptus development in pigs. [supported by the Next Generation BioGreen 21 Program (#PJ01110301), Rural Development Administration]

Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.

Allicin (AL) is one of the biologically active substance in garlic. Many researchers found that AL exhibits 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 development. The oocyte was maturated in vitro for 44 h (control) without AL or 44+24 h IVM (24 h aging) with 0, 0.1, 1, 10 and 100 μM allicin (0 AL, 0.1 AL, 1 AL, 10 AL and 100 AL). The 1 AL treated group was significantly increased on maturation rate compared to the 0 AL treated groups, but the other treated groups were not different compared to the 0 AL treated group (p < 0.05). The 1 AL treated group was significantly increased on normal spindle formation and chromosome alignment compared to 0 AL treated group. We checked the effect of AL on parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development using aged oocytes. PA embryos in 1 AL treated group significantly increased the cleavage and blastocyst formation rate compared to control (p < 0.05). However, SCNT embryos of 1 AL treated group were no significant differences in embryo development. In PA embryo quality, the total cell number was significantly higher in 0.1 AL, 1 AL and 10 AL treated groups than control and DNA fragmentation rate in 1 AL was the lowest among all groups. SCNT embryo in 1 AL significantly increased total cell number and decreased DNA fragmentation. The AL treatment on aged oocyte enhanced PA and SCNT embryo developmental capacity. Therefore, AL may be helpful for assisted reproductive technology applying aged oocytes.

Maturation-promoting factor (MPF) is well-known as cell cycle regulator during oocyte maturation and fertilization. MPF activity maintains high levels and arrest the cell cycle progression until fertilization. After fertilization, Anaphase-promoting complex/cyclosome (APC/C) mediated degradation of cyclin B causes decrease of MPF activity. One of the cytostatic factor (CSF), Emi2 inhibits APC/C activity by binding to APC/C-cdc20, therefore blocks the proteolysis of cyclin B. Degradation of Emi2 requires phosphorylation by Polo-like kinase 1 (Plk1). Thus recognition and phosphorylation of Emi2 by Plk1 are essential step for meiotic cell cycle resumption. In our previous research, we found that two phosphorylated threonine regions at amino acid position 152 and 176 in Emi2 are respectively contributed for recognition by polo-box domain of Plk1. Peptidomimetics 103-8 can block the interaction between Plk1-PBD and Emi2, and therefore meiotic maturation and meiosis resumption via parthenogenetic activation were impaired. However, major drawback of 103-8 was the limitation of penetration through the cell membrane. We synthesized the new peptidomimetics and checked bioavailability in mammalian oocyte by injection and media treatment. Medium treatment with peptidomimetics C-4, meiotic maturation has significantly decreased and meiotic resumption via parthenogenetic activation has perfectly impaired. For the next experiment, we are preparing X-ray crystallography to identify the binding modes between Plk1-PBD and peptidomimetics C-4.

In pig, more than half of the recovered cumulus cell-oocyte complexes (COCs) have one or two layers of cumulus cells and are considered morphologically poor. If we could take full advantage of these poor quality COCs, we could potentially improve the efficiency of in vitro embryo production. During in vitro maturation, although some maturation factors are transmitted bidirectionally between the oocyte and cumulus cells of the same COC, transmission also occurs between different COCs. We hypothesized that morphologically poor COCs fail to undergo complete oocyte maturation due to their insufficient secretion of maturation factors. Here, we investigated whether co-culture with morphologically good COCs (having three or more layers of cumulus cells) could improve the maturation and utilization rates of morphologically poor COCs. Our results revealed that the oocyte maturation rate, glutathione level, embryo development capacity, blastocyst quality, and cumulus cell gene expression levels of BCL-2 and PCNA were similar in the co-culture and good quality-groups, and that these levels were all significantly higher than those in the poor quality-group. Our results strongly suggest that the co-culture strategy greatly improved the utilization rate of morphologically poor COCs without reducing their capacity for maturation and subsequent development.

α-solanine is toxic to human health by disturbing digestive and central nervous systems. However, little information has been focused on investigated with respect to α-solanine influence in mammal oocyte maturation and quality. In this study, we investigated the effects of α-solanine on oocyte maturation, quality and possible molecular mechanisms in a pig model. Porcine Cumulus-oocyte complexes (COCs) were treated with increasing concentration (0, 1, 10, 20, 50 μM) of α-solanine subjected to further in vitro maturation culture. The result showed that α-solanine significantly inhibited cumulus cells expansion and increased oocyte death rates when the concentration of α-solanine more than 10 μM. After cell cycle and cytoskeleton analysis, the results showed that α-solanine (10 μM) disturbed meiotic resumption, increased abnormal spindle formation and cortical granules (CGs) distribution rates when compared with the untreated group. α-solanine (10 μM) triggered autophagy by increasing the expression of autophagy-related genes (LC3, ATG7, LAMP2) and accumulation of LC3-specific puncta (an autophagy maker). TUNEL staining assay showed that α-solanine significantly increased apoptosis in porcine oocytes confirmed by up-regulated the levels of BAX and CAPS3 genes. Further study revealed that exposure α-solanine (10 μM) to porcine oocytes induced ROS generation, reduced mitochondrial membrane potential. In addition, our results suggested that α-solanine (10 μM) significantly increased the levels of H3K36me3 and H3K27me3 in porcine oocytes. Taken together, these data indicated that α-solanine toxic impaired oocyte maturation and quality by inhibited cumulus cells expansion, increased abnormal spindle and CGs distribution rates, triggered autophagy/apoptosis occur, accumulated ROS, decreased mitochondrial membrane potential, and changed epigenetic modifications.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM, and protein kinase B (PKB) expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was treated in maturation medium (TCM199) while process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated each group of oocytes and CCs were sampled. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR was performed. In CCs, all of GDF8 treatment groups showed significantly higher CREB transcription regulator cbp mRNA and the 1- and 10 ng/ml treatment groups observed significantly increased cumulus expansion related genes areg, cox-2, has2, ptx3 and tnfaip6 transcription levels after IVM. In matured oocytes, the maternal factors jmjd3 and zar1, transcriptional regulator foxo1 and sirt1, mitochondrial activity factor sirt3 and acadl, and anti-apoptosis gene bcl-2 mRNA transcription levels were significantly increased in 1- and10 ng/mL of GDF8 treatment groups compared with control. To determine effect of GDF8 treatment during IVM, translation regulator PKB protein expression and phosphorylation levels were analyzed in CCs by western blotting. The 10 ng/ml treatment group showed significantly increased phosphorylated PKB (1.4 times higher than control) protein levels (P < 0.05). In conclusion, treatment 10 ng/ml of GDF8 during IVM activates CREB related transcription and induced cumulus cells expansion via activation of PKB signaling in CCs. The transcriptional landscape changes in CCs result maternal factors accumulation and mitochondrial activation in oocytes during IVM.

Mitotic spindle formation is regulated by centrosomes, composed of a centriole pair surrounded by pericentriolar materials(PCM) proteins. However, mammalian oocytes rely on acentriolar MTOCs for the function of meiotic spindle. The composition of acentriolar MTOCs and the molecular precesses that regulate the localization and accumulation in mammalian oocyte are not well understood. In this study, we analyzed the mechanisms of spindle microtubule nucleation and stability from MTOCs in mouse oocyte, and indentified Centrosomal protein192(CEP192) as a key regulator for acentriolar MTOC formation. CEP192 specifically colocalized with pericentrin (PCNT) during the oocyte maturaion. CEP192 proteins are localized throughout cytoplasm and around nucleus at GV stage, and then after BD stage, CEP192 proteins were further fragmented into smaller MTOCs around chromosomes. At metaphase, CEP192 proteins were concentrated in spindle pole. Knockdown of CEP192 using siRNAs resulted in metaphase I arrest. The arrested oocytes were characterized by reduced microtubule intensity and misalignment chromosome. Also at BD and ProMI stage, the oocytes reduced microtubule density and PCNT intensity. To confirm the mechanism of CEP192 regulation, we confirmed that PLK1 and AuroraA kinase were involved in CEP192 activation. The investigations for detailed molecular mechanisms of CEP192 and RanGTP for microtubule nucleation in oocytes are underway using various techniques including siRNA, mRNA, and positive or negative dominant injection and inhibitors.

Melatonin has an important role as anti-oxidative effect and reducing of endoplasmic reticulum(ER)-stress on oocyte maturation and embryo development. Under ER-stress condition, unfolding protein response (UPR) is a defence mechanism in mammalian cells. Recently, regulation of UPR signaling genes are involved in oocyte maturation, embryo development and female reproduction. However, there is no report on the role of melatonin for UPR signaling and ER-stress mediated apoptosis during pig oocyte maturation progression. Moreover, the changes of UPR genes expression according to the porcine oocyte maturation is not yet fully understood. Here, we investigated the changes of UPR signal (BIP/GRP78, ATF4, p90/p50ATF6, and XBP1) and ER-stress apoptotic factor CHOP genes expressions in porcine oocyte maturation by Western blot and RT-PCR analysis. During oocyte maturation, UPR marker and CHOP genes expressions were significantly increased in matured oocytes or cumulus-oocyte complexes (COCs). UPR markers expressions were significantly increased by ER-stress inducer, tunicamycin (Tm), treated (1, 5, 10 μg/ml) groups in a dose-dependent manner compared with control group. To confirm the reducing of ER-stress by melatonin (0.1 μM), we were compared to the effects of ER-stress inhibitor, TUDCA (200 μM), after pre-treated Tm (5 μg/ml) for 22 h maturation. Expressions of UPR markers and meiotic maturation were recovered by melatonin (0.1 μM) in COCs. And, we observed the role of Grp78/Bip as UPR signaling beginning marker using siRNA. In result, reduction of Grp78/Bip gene expression by siRNA was induced the inhibition of oocyte maturation (32.5±10.1 vs control; 77.8±5.3), and p50ATF6 protein level was significantly decreased (p<0.001) in cultured COCs for 44 h. In addition, these results were recovered through the addition of melatonin (0.1 μM) or TUDCA (200 μM) in maturation medium. These results demonstrated that the regulation of UPR signaling via Grp78/Bip gene induction plays a critical role in porcine oocyte maturation in vitro. Furthermore, this present study first confirmed a functional link between inhibition effect of ER-stress by melatonin and regulating of UPR signaling in porcine oocyte maturation. In conclusion, melatonin improves the oocyte maturation and cumulus cells expansion of COCs through the regulation of UPR signal pathway by BIP/GRP78 against the ER-stress during porcine oocyte maturation periods.

The plastic monomer bisphenol A (BPA) is well known as a representative environmental hormones. Recent studies showed that the BPA exposure induced mitochondrial dysfunction and mitochondrial derived reactive oxygen species (mito-ROS). However, changes of antioxidant enzymes expression and ROS production from mitochondria according to the BPA exposure on in vitro maturation (IVM) of porcine oocytes have not been studied. We hypothesized that regulation of ROS production from mitochondria by BPA may play a critical role in meiotic maturation or expansion of cumulus cells in cumulus-oocyte complexes (COCs). To investigate the negative effects of BPA exposure on oocyte maturation, immature pig oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75 and 100 μM) for 44 h. Expectedly, the rates of meiotic maturation and cumulus cell expansion of COCs in the BPA (75 μM) treated group was significantly lower than those of control group (p<0.01). Most of secretion factors expressions from COCs were significantly decreased (p<0.05) in the BPA treated COCs. Next, we investigated the intracellular ROS and mitochondrial specific superoxide production according to the BPA exposure using DCF-DA and mito-SOX staining, respectively. BPA exposure were showed that increasing of both intracellular ROS and mito-ROS, as well as mitochondrial related antioxidant enzymes (sod2, prdx3, prdx5) mRNA expression significantly increased (p<0.01) in COCs. And then, mitochondria membrane potential (MMP) dramatically reduced, and mitochondrial-derived apoptotic factors (bax, bcl-xl, caspase 3) mRNA expressions were increased (p<0.01) in BPA treated COCs. In additon, protein levels of mitochondrial-derived apoptosis genes (AIF, cleaved parp1 and caspase 3) were significantly increased (p<0.05) by BPA exposure. To confirm the reduction of BPA-induced mito-ROS, we used to the mitochondrial-targeted ROS scavenger, mito-TEMPO. Interestingly, addition of mito-TEMPO (0.1 μM) to the BPA pre-treated COCs recovered in meiotic maturation of porcine oocytes. These results demonstrated that BPA exposure was induced increasing of mitochondrial dysfunction, mito-ROS and mitochondrial-mediated apoptosis on pig oocyte maturation. Therefore, we suggest that controlling of mito-ROS plays a critical role in pig oocyte maturation in vitro. These findings will be helpful to solve causes of mitochondrial-related infertility.