The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

In mammal, oocytes are arrested at the metaphase Ⅱ until fertilization. However, unfertilized oocytes that remain in the oviduct or under in vitro culture, which is called "oocyte aging". Asynchrony negatively affects fertilization, pre- and post-implantation embryo development. Caffeine is known to phosphodiesterase inhibitor that rescues oocyte aging in several species. Nevertheless, the effect of caffeine was not clear in bovine aging oocytes. In this study investigated the cytoskeleton distribution in aged oocytes and the embryo development ability of aged oocytes from treated with or without caffeine during maturation. The cumulus and oocyte complexes (COCs) were cultured in 10% FBSTCM199 for up to 22h at 38.5℃ in 5% CO₂. For oocyte aging study, the COCs were cultured in 10% FBS-TCM199 supplemented with or without 10 mM caffeine for 40hs. And then oocytes underwent in vitro fertilization using highly motile sperm recovered from frozen and than thawed bull semen. As a result normal cytoskeleton percentage of caffeine treatment group more than the aging group (67.57%±4.11 VS 44.61%±6.40) and no significantly different compared to control group. Aged oocytes derived from addition of caffeine to the in vitro maturation medium significantly increased the percentage of 2- cell that developed to the blastocyst stage compared to the aging group. Blastocysts derived from caffeine treatment group significantly increased the total cell number compare aging (90.44%±10.18 VS 67.88%±7.72). Apoptotic fragmenting of genomic DNA was measured in individual embryos using the TUNEL assay. Blastocyst derived from caffeine treatment group significantly decereased the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocytes aging periode can improved the develpmental rate and quaility in bovine embryos developing in vitro.

Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In this report, we examined the cytotoxic effects of chitosan nanoparticles on mouse embryos at the blastocyst stage and in vivo implantation by embryo transfer. Blastocysts treated with 250 nm chitosan nanoparticles exhibited significantly increased apoptosis and a corresponding decrease in total cell number, which was concentration‐dependent. Moreover, the TUNEL positive signal in the embryos exposed to chitosan nanoparticles showed an increased of the ICM and the implantation success rate was lower than that of their control counterparts. Our results collectively indicate that in vitro exposure to chitosan nanoparticles induces apoptosis and retards implantation development after transfer to host mice. The results collectively show that chitosan nanoparticles have the potential to induce embryo cytotoxicity. Further studies are required to establish effective protection strategies against the cytotoxic effects of these nanoparticles.

Semen can be divided into two parts. One is cellular part which contains sperms the other is liquid part which is called by seminal plasma. The seminal plasma is a nutritive and protective medium for the sperms. Fructose, which is major energy source, is supplied to sperms swim to female oocyte. Alkalic property protects sperms from hostile environment of female reproductive organ. Also, seminal plasma induces tolerance to preexisted immune cells, and changes intra‐uterine environment to better conditions for fertilized embryos to implant. However, the effects of seminal plasma in in vitro culture of fertilized embryos are unclear. Second fraction of fresh semen was obtained from a normal farm pig. The semen was centrifuged to remove sperms, and then supernatant was filtrated. The filtered seminal plasma was stored in — 30℃. In this study, electrically activated and chemically activated porcine embryos were employed to investigate the developmental rate after 2 hours treatment of none, 0.1%, 0.5%, and 1% seminal plasma in culture media by two days of activation. Both electrically and chemically activated embryos, cleavage rate and cell numbers of blastocysts were not significant difference within four groups. Blastocyst formation rate of electrically activated embryos also did not show significant difference within any groups. However 0.1% seminal plasma treatment group showed significantly increase of blastocyst formation rate in chemically activated group (None; 24.8%, 0.1%; 31.7%, 0.5%; 19.4, and 1%; 16.5%, respectively. p<0.05).

It is well established that mammalian cumulus cell (CC) expansion requires BMP15 (bone morphogenetic protein bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9). However, the mechanisms of the factors in CC expansion are largely unclear. This study was conducted to examine the two paracrine factors and their receptor SMAD intracellular signaling mechanism of mediating porcine CC expansion and oocyte maturation, and to compare COCs (Cumulus–oocyte complexes) maturation to DOs (Denuded oocytes). COCs and DOs were in vitro matured in medium with FSH, LH and TGFB superfamily antagonists. Our results showed that the expansion of COCs was unaffected by addition of GDF9 and BMP15 recombinant protein, but cumulus cell proliferation and DOs maturation rate were enhanced. The mRNA expressions of SMAD receptor confirmed that oocytes secreted factors that activate SMAD3,4 and SMAD1 in granulosa cells and oocytes, but unaffected SMAD2. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) inhibited CC expansion and expression of TNFAIP6. SB431542 also was revealed to inhibit DOs maturation. The activation of CC SMAD signaling by oocytes, and the requirement of SMAD2/3 signaling for expansion and oocyte maturation were studied in pig. Nonetheless, porcine oocyte maturation without SMAD2/3 signaling is likely to be needed for optimal matrix formation, but also BMP15 and GDF9 is likely to be needed in oocyte.

In the present study, we investigated the effect of porcine follicular fluid (PFF) concentration (10% vs. 1%) and protein-free media (PFF 0%) on maturation of porcine oocytes in vitro and analysed difference in gene expression in resulting blastocysts following parthenogenetic activation. Three groups were tested; 1) 10% PFF: Tissue culture medium (TCM) 199+10% PFF; 2) 1% PFF: TCM 199+1% PFF; and 3) 0.1% PVA: TCM 199+0.1 PVA. Cumulus-oocyte-complexes were cultured in the respective media containing gonadotrophin (1 ug/ml), epidermal growth factor (10 ng/ml), cystein (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 ug/ml), 9-cis retinoic acid (5 nM) for 20～22 h and then without hormonal supplements for an additional 20-22 h. Data was analyzed using statistical analysis system(SAS) program. There was no significant difference in oocyte maturation rate. However, significantly higher (p<0.05) proportions of embryos developed to the blastocyst stage when oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 vs. 54.6±3.1 vs. 54.4±2.5, respectively). The relative abundance (ratio to beta-actin mRNA) of gene transcripts related to apoptosis in blastocysts was measured by real- time PCR. The expression of anti-apoptotic gene (BclxL) was up-regulated and the expression of pro-apoptotic gene (Bax) was down-regulated in 10% PFF group than in the other groups. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by reduction of apoptosis. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), MKE (#10033839-2011-13), Institute for Veterinary Science, the BK21 program and TS Corporation.

The present study examined the expression of porcine sirtuin 1–3 (Sirt1–3) genes in immature (germinal vesicle; GV stage), mature (metaphase II; MII stage) oocytes, preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the role of sirtuins in oocyte nuclear and cytoplasmic maturation, and embryonic development of PA and IVF embryos using sirtuin inhibitor [5 mM nicotinamide (NAM) and 100 μM sirtinol]. The expression of Sirt1–3 mRNA was significantly (p<0.05) up-regulated during IVM. The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF and SCNT were gradually (p<0.05) decreased from MII stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1 and Sirt3 in SCNT blastocysts were significantly lower than IVF blastocysts. Treatment with nicotinamide (NAM) during IVM resulted in significantly decreased nuclear maturation but it was restored when NAM treated with 2 μM resveratrol (RES; known as antioxidant and sirtuin activator) compared to the control (control: 88.9%, NAM: 67.9% and NAM+RES: 86.4% respectively). Intracellular reactive oxygen species (ROS) level of oocytes matured with NAM was significantly increased and with NAM+RES was significantly decreased compared to the control. Treatment with sirtuin inhibitors during IVC resulted in significantly decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate both PA and IVF embryos. Oocytes treated with NAM during IVM showed significantly lower expression of PCNA, Bax, Bcl-2, POU5F1 and Sirt1–3 compared to the control. Oocytes treated with NAM+RES during IVM restored gene expression except POU5F1. Similarly, PA derived blastocysts treated with NAM during IVM showed down-regulation of PCNA, Bax, Bcl–2, POU5F1 and Sirt1–2. The blastocysts derived from PA embryos treated with sirtuin inhibitors during IVC showed lower (p<0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly de -creased in both NAM and sirtinol treated groups compared to the control. These findings indicate that Sirt1–3 which are transcribed and stored during oocyte maturation may have physiological and important roles in porcine oocyte maturation and preimplantation embryonic development by regulating gene expressions. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The objective of the present study was to investigate the effects of different concentrations of sorbitol supplementation for in vitro maturation medium and in vitro culture medium, on porcine cumulus oocyte complexe(COC) maturation and subsequent developmental capacity after parthenogenetic activation. Porcine COC were cultured for 44 h(0～ 22 h termed MI stage and 22～44 h termed MII stage) in TCM199 without(— ) or with(+) sorbitol (20 μM, 100 μM, 200 μM), and the resultant metaphase II oocytes cultured in PZM-3 for 7 days following activation. Our results showed that supplementation with appropriate concentrations of sorbitol (20 μM) during full term maturation culture(MI+/MII+) significantly(p<0.05) improved blastocyst formation rates and total cell number. When the concentration of sorbitol were increased to 100 μM and 200 μM during maturation culture, the maturation rate of COC were significantly reduced compared with 20 μΜ or control groups. Also blastocyst formation rates significantly(p<0.05) reduced with increasing concentration of sorbitol(200 μM). Supplementation with sorbitol(20 μM, 50 μM, 100 μM) into PZM-3 for in vitro culture significantly(p<0.05) inhibited blastocyst formation compared with control group. However, the blastocyst formation rates start to rise again when 50 μ M sorbitol was used for the first 48 hours and then cultured in PZM-3 without sorbitol. There was no significant difference in cell number between control and sorbitol treated groups. When the activated oocytes were cultured in PZM-3 for 48h and then cultured in PZM-3 with sorbitol, interestingly, the blastocyst formation rate was similar to that of PZM-3 with sorbitol for in vitro culture and significantly lower than control group. These results suggest that addition of low concentrations of sorbitol(20 μM) during oocyte maturation is beneficial for subsequent blastocyst development and improved embryo quality. However, treatment with sorbitol supplementation during in vitro culture medium is negative effect to blastocyst formation.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

191개의 아미노산으로 구성된 인간 성장 호르몬(human Growth hormone, hGH)은 성장 촉진뿐만 아니라 근육량 증가, 뼈 강화, 체내 지방 분해 등의 약리적 작용을 가지며, 이와 연관된 여러 질환에 대한 치료 및 기타 치료보조제(미용 및 노화억제 분야 등)로 사용되고 있다. hGH를 비롯한 대부분의 단백질 치료제는 98% 이상이 주사제로 투여되고 있는데 이 러한 투여 방식은 환자의 통증 및 감염 가능성 뿐만 아니라 투여 횟수가 많은 경우에는 시 간적, 비용적, 편리성의 측면에서 환자에게 부담이 가중된다. 본 연구에서는 이러한 문제점 을 해결하기 위하여 감염의 우려도 없으며 주사공포증 없이 복용할 수 있는 hGH와 hTf 단 백질을 융합시킨 형태의 경구 투여용 hGH를 생산하고자 하였다. hGH와 hTf 융합 단백질의 유전자 서열은 HepG2 세포에서 분리한 RNA로부터 RT-PCR 을 수행하여 cloning한 hTf 유전자의 서열과 cDNA로 합성한 hGH 유전자 서열을 cyclo peptide linker나 helical peptide linker로 연결하여 retrovirus vector에 도입하였다. 구축한 각 virus vector는 virus 생산 세포주인 GP2 293과 VSV-G 피막단백질 유전자를 이용하여 retrovirus로 생산한 후, 닭의 배아섬유아세포인 CEF와 CHO 세포에 감염시켰다. 각 세포에서 hGH-hTf 유전자의 발현은 RT-PCR, Western blot, ELISA 실험을 통하여 확인하였다. RT-PCR 실험 결과에서는 virus에 감염된 세포주와 감염되지 않은 세포주에서 GAPDH 유전자에 대한 증폭 단편이 확인된 데 반해, hGH 유전자와 WPRE 서열에 대한 증폭은 virus 에 감염된 세포주에서만 이루어 졌다. Virus에 감염된 세포에서 hGH 단백질과 hTf 단백 질의 발현 양상을 확인하기 위하여 각각의 세포 배양액과 세포에서 분리한 단백질을 이용 하여 Western blot을 실시하였다. 세포 배양액과 세포에서 분리한 단백질에서의 hGH와 hTf 발현을 비교한 결과, 두 단백질 모두 세포 배양액에서의 발현이 강한 것으로 확인되었다. hGH 단백질은 약 20 kD의 크기를 나타내었으며 hTf 단백질은 80 kD의 크기를 가지는 것 으로 확인되었다. 각 virus에 감염된 세포에서는 hGH 단백질이 hTf 단백질과 융합된 형태 로 발현되어 약 100 kD의 크기를 가지는 것으로 확인되었다. ELISA 분석 실험에서도 virus 에 감염된 각 세포에서의 hGH 단백질의 발현 및 분비를 확인하였다. 본 연구에서 확보한 경구 투여용 인간 성장 호르몬인 hGH-hTf는 차후 형질전환 동물의 개발이나 물질 생산 세포주의 확립을 통해서 대량생산함으로써 주사용으로만 개발되어 있 는 바이오의약품의 경구용화 관련 연구에 필요한 핵심 기술을 제공할 수 있을 것이다. * 본 연구는 농촌진흥청 차세대 바이오그린21사업(과제번호: PJ00804101)의 지원에 의해 이루어졌다.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

In ruminants, Interferon-τ (IFN-τ) has the role of recognizing pregnancy signals produced by the embryo and it may have an important role during the luteolysis. Therefore, the purpose of the present study was to investigate the effect of IFN-τ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and secretion of progesterone (P4) in vivo. The epithelial and stromal cells isolated from bovine endometrium were cultured with different doses of IFN-τ (0, 0.02, 0.2 and 2 μg/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin E2 and F2α levels in the culture media were analyzed by enzyme immunoassays, and total RNA was extracted from the cells for RT-PCR. P4 concentrations in blood samples were assayed by chemiluminescent immunoassay system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-τ (p<0.05), but it was not significantly different in all groups of stromal cells except 2 μg/ml IFN-τ group (p<0.05). Although IFN-τ did not affect PGE2 and PGF2α production in epithelial cells, it decreased PGE2 and PGF2α production significantly in stromal cells (p<0.05). In vivo experiment, the P4 concentrations in blood sample was significantly increased after injection of 1 μg/ml IFN-τ. These results indicate that PG production was mediated by COX-2 expression in the stromal cells but it did not affect in the epithelial cells, and suggest that treatment of IFN-τ was to improve the implantation environment of uterine by maintenance of high P4 concentration. * This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ907008)” Rural Development Administration, Republic of Korea.

본 시험에서 건초(티머시, 알팔파 및 클라인)와 짚류(톨페스큐 및 볏짚)의 buffer 용해도와 단백질 분획이 실시되었으며, 조사료 자원의 buffer 추출이 in vitro 발효 성상, 분해율 및가스(CO2 및 CH4) 생성량에 미치는 효과를 조사하였다. 다른 조사료에 비해 총 단백질 중buffer 가용성 조단백질과 A fraction은 알팔파건초에서 각각 61% 및 41.77%로 가장 높았으며 볏짚에서 가장 낮았다(각각 42.8% 및19.78%). 총단백질 중 B1 fraction은 조사된 조사료간 비교적 큰 차이를 보이지 않았으나 B2fraction에서는 다른 조사료(6.34~8.85%)에 비하여 톨페스큐짚(10.05%) 및 클라인 건초(12.34)%에서 다소 높은 수준을 보였다. 총 단백질 중B3 fraction이 차지하는 비율은 톨페스큐짚에서38.49%로 가장 높았으나 다른 조사료 자원 간에는 큰 차이가 없었으며, C fraction의 경우 볏짚에서 가장 높은 비율(15.05%)을 보였다. 모든 사료에서 배양 개시 후 3시간(P<0.01) 및 6시간(P<0.05)에서 buffer 추출 전에 비해 추출후 배양액의 pH가 증가되었으며, 배양 6시간(P<0.05) 및 12시간(P<0.001)에서 다른 사료에비해 티모시 건초 및 알팔파 건초로부터의 pH가 낮았다. 배양액의 암모니아 농도는 모든 배양시간에서 가용성 물질의 추출 전 후에 다른조사료에 비해 알팔파 건초에서 가장 높았으나모든 사료의 추출효과는 배양 3시간(P<0.01)에서만 나타났다. 배양액의 총 VFA 농도는 배양24시간까지 알팔파 건초에서 가장 높았던 반면톨페스큐짚과 볏짚에서 가장 낮았다. 또한 모든 조사료에서 buffer 추출 전에 비하여 추출후에 총 VFA 농도가 감소되었다(P<0.01~P<0.001). Acetic acid (C2)의 조성 비율에서는 배양 6시간까지 추출 전에 더 높았으나(P<0.001)사료 간 차이는 없었다. Propionic acid (C3) 조성 비율 역시 배양 개시 후 3, 24 및 48시간(P<0.001)에 추출 전에 더 높았으며, 6 및 12시간의 배양액에서 대부분 건초(티모시, 알팔파 및 클라인)와 짚류(톨페스큐짚 및 볏짚) 간차이가 있는 것으로 조사되었다(P<0.05). 그러나 butyric acid (C4) 조성비율의 경우 대부분의배양시간에서 사료 간 차이는 없었다. 건물에서의 분해율 관련 parameter 중 a 값은 조사된전체 조사료에서 buffer 추출 전이 추출 후에비해서 높았으며(P<0.001), 다른 조사료에 비해톨페스큐짚과 볏짚에서 크게 낮았다(P<0.05).또한 b 값의 경우 역시 추출 전에 비해 추출후에서 현저히 낮았으나(P<0.001) 사료 간 차이는 없었다. 볏짚을 제외한 조사료에서 추출후에 비해 추출 전의 건물 유효분해율(EDDM)이 더 높았다(P<0.001). 조단백질에서의 a, b및 c 값은 추출 전에 비해 추출 후에서 현저히낮았으나(P<0.05) 사료 간 차이는 없었다. 조단백질 유효분해율(EDCP)에서는 다른 조사료 종류에 비해 톨페스큐짚과 볏짚에서 낮았다(P<0.05). 한편, NDF의 경우 a 값과 b 값(P<0.01)및 NDF 유효분해율(EDNDF, P<0.001)은 추출후에 비해 추출 전에 더 높았으나(P<0.01) 사료 간 차이는 보이지 않았다. 반추위미생물에의해 사료분해과정 중 생성되는 CO2 량도 24시간 배양까지는 추출 전에 더 많았으며(P<0.05~P<0.001), 톨페스큐짚과 볏짚에 비해 건초형태의 조사료로부터의 CO2 생성량이 더 많았다(P<0.05~P<0.01). 메탄가스(CH4) 생성량 역시 모든 배양시간에서 추출 전에 비해 추출 후에 크게 감소되었으며(P<0.01~P<0.001), 12~24시간을 제외하고는 짚류에 비해 건초에서 현저히 높은(P<0.05) 것으로 나타났다. 본 시험의 결과를 종합하면, 조사료 자원에 대한 buffer용해도와 단백질의 분획이 in vitro VFA 농도와 분해율 및 gas (CO2 및 CH4) 발생량 간 상호 밀접한 관계를 보이는 것으로 여겨진다. 이에 따라 조사료 이용 효율 개선을 위해 조사료자원에 대한 buffer 용해도와 단백질 분획을 반추동물 TMR 조제에 활용할 필요가 있는 것으로 여겨진다.

The objective of this study was to examine the effect of thymidine treatment during in vitro maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group (p<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group (p<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group (p<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups (p<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

본 연구는 국내에서 육성된 거베라 ‘Saebom’, ‘Songsongee’ 그리고 ‘Sugar Pink’ 등 3품종을 이용 하여 LED와 cytokinin이 기내 유묘의 생장 및 형태 형성에 미치는 영향을 조사하기 위하여 수행하였다. MS 기본배지에 BAP 0, 1.0, 2.0, 4.0mg·L-1와 kinetin 1.0, 2.0, 4.0 mg·L-1 각각에 NAA 0.2 mg·L-1를 혼용하 고, LED는 적색, 청색, 혼합(적색+청색) 및 형광등으 로 하여 배양 6주 후 기내 유묘의 생육을 조사하였다. 3품종 모두 신초 생육에서 신초장은 대조구인 형광등 보다는 적색광 처리구에서 길어지는 경향이었으며 엽 신은 작아지고 엽병은 가늘어지는 형태를 보였다. 특히 BAP에서의 신초는 대부분 로제트 타입으로 생육하였다. 신초수는 형광등과 혼합광 처리구의 BAP 4.0 mg·L-1 에서 가장 많았고, BAP가 kinetin보다 신초수 증가에 더 효과적이었으나 투명화율이 높게 나타났다. 뿌리의 생육은 3품종 모두 모든 광원에서 cytokinin 무처리구 에서 양호하였다. 또한 생체중과 건물중 그리고 엽면적 값은 혼합광 처리구에서 높은 경향을 보였다. 엽록소 함량은 ‘Songsongee’ 품종을 제외하고는 청색광 처리 구에서 높게 나타났으며 3품종 모두 조사된 모든 광원 에서 kinetin이 BAP보다 더 높은 엽록소 함량을 보였 다. 투명화율의 경우는 모든 광원에서 BAP의 농도가 증가할수록 높아지는 경향을 보였으며 kinetin 처리구 에서는 투명화가 거의 나타나지 않았다.

In the present study, the effect of cysteine and NT or bisphenol A (BP) on in vitro aturation (IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% CO2 and 95% air at 38℃. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5～10.0 mM cysteine were 34.0±3.2%, 36.0±3.5%, 48.0±3.8%, 22.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5～5.0 mM NT for 48 hrs were 24.0±4.2%, 18.0±4.9%, 8.0±2.2%, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine (38.0±4.3%) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05～5.0 mM BP for 48 hrs were 20.0±4.7%, 10.0±5.3%, 6.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP (44.0±3.5%). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cysteine (32.0±3.2%) were increased than that of BP treatment.

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03～0.10 mM SN and 0.3～1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a CO2 incubator (5% CO2, 95% air, 38℃). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were 25.9±3.5%, 36.4±3.2%, 33.3±3.5%, 28.8±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03～0.07 mM SN were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were 28.0±4.2%, 36.5± 3.6%, 30.0±3.8%, 19.2±3.5%, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.05 mM SN were 26.0±3.2%, 28.0±3.4%, 38.0±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.5 mM NO were 22.0±3.0%, 30.0±3.8%, 36.0±4.2%, respectively. These result was significantly increased compare to the control.

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.