돼지 핵이식 복제수정란의 체외 발달율을 개선하고자, 핵이 주입된 수핵난자를 전기자극 에 의한 융합 후 demecolcine으로 세포질 활성화 처리를 실시하였다. 2-세포기로 분할 전 까지 핵의 이상분열을 억제하여 정상적인 핵형 유지여부 및 demecolcine이 핵의 방출을 유 도하여 탈핵의 전 처리, 핵이식 후 활성화의 후 처리 및 탈핵 전․후 처리를 모두 한 핵이 식 수정란의 체외발달율을 조사하였다. 융합이 이루어진 복제 수정란에 demecolcine을 4시간 처리하였을 때 정상적인 핵 모양인 CLC(clustered chromosomes) 상태는 43.2%로서 무 처리구의 15.0%보다는 높았으며, 1PN (single pronucleus) 상태는 46.9%로서 대조구의 35.0%와는 차이가 없었다. 비정상적인 핵 형인 ≥2PN, PPB+CLC or PN 및 ≥2PN 7.4, 0 및 2.5%로서 무 처리구의 17.5, 22.5 및 10%로서 처리구가 낮았다. 융합 이후 16시간에는 1PN이 85.0%로서 대조구의 56.7%보다 는 높았다. 분할율은 전․후 모두 처리구에서 86.4±2%로서 후 처리(81.3±6%)와 대조구(79.6±6%) 보 다 높았으며, 전 처리의 85.7±2%와는 차이가 없었다. 배반포기로의 발달율에 있어서도 전․후 처리가 18.1±3%로서 전 처리(11.0±3%)와 대조구(11.3±1%)에 비하여 높았으나, 후 처리의 15.5±1%와는 차이가 없었다. 배반포기 수정란의 할구수도 전․후 처리가 21.6±6% 로서 전, 후 처리 및 대조구의 각각 18.8±8%, 19.4±3% 및 19.7±2%보다 많았다. Demecolcine을 돼지 복제 수정란에 처리하면 분할 전까지 핵의 이상 분열을 억제하는 것 이 확인되었으며, demecolcine 처리에 의한 탈핵은 수핵난자의 세포질 손상을 감소시키고 보다 용이하게 할 수 있다. 본 연구에서와 같이 탈핵 및 활성화를 목적으로 탈핵 전․후 처 리를 하였을 때 체외발달율이 다소 개선되는 경향을 보였다.

소의 수정란 이식기술은 우수한 유전 형질을 가진 개체를 효과적으로 증식 시킬 수 있고, 형질이 동일한 다수의 자축을 단시간 내에 생산이 가능하므로 가축의 능력개량에 매우 유 용하게 이용할 수 있다. 최근, 일시적인 저영양 처리가 호르몬제 투여에 의한 과배란 유도 에 앞서, 한우 공란우 체내수정란 생산효율을 향상시킨다는 보고가 있다. 본 연구는 국립축 산과학원 가축유전자원 시험장에서 사양.관리 중인 한우를 공시동물(53두)를 이용했다. 총 16번의 과배란유도 처리를 시행하였고, CIDR삽입 1주전부터 채란 및 이식까지 23일간 번 식우 사양프로그램에 기준해 대조구(19두)는 배합사료 1 kg, 고영양구(13두)는 2.5 kg 그리 고 저영양구(21두)는 0 kg으로 일시적인 제한급여를 실시하고, 조사료는 자유채식을 하였 다. 과배란유도 처리는 공란우의 발정주기에 관계없이 CIDR삽입 0일을 기준으로 7일 전부 터 영양처리에 제한을 주고, 4일째부터 4일간 FSH를 근육주사 하였다. 그리고 투여 6일째 CIDR제거와 동시에 PGF2α를 오전 5 mL, 오후 3 mL를 근육주사 하여 과배란을 유도하였 다. 한편, 공란우의 인공수정(AI)은 8～9일째 12시간 간격으로 정액 2straw당 각각 2회 AI 를 실시하였으며, 16일째 비 외과적 방법(자궁관류법)으로 채란하였다. IETS 수정란 등급판 정 기준에 따라 CODE 1, CODE 2를 이식가능 수정란으로 판정하였다. 공란우 53두를 과 배란 처리 하여 Estrogen과 Progesterone의 농도를 분석한 결과, Estrogen의 경우 고영양 에 비해 저영양에서의 호르몬수준이 CIDR삽입 일에 30 IU 정도 더 높았다. 이 결과로부터 일시적인 저영양이 발육중인 난포수의 증가에 영향을 미침을 알 수 있었다. 한편, Progesterone 분비량은 두 처리구 간에 유의적인 차이는 없었지만, 수정 후 Progesterone의 농도 의 증가로부터 정상적으로 황체호르몬이 분비되었음을 알 수 있었다. 일시적인 영양처리에 따른 체내수정란 회수난 수와 이식가능 수정란 수(%)는 각각 대조구에서 8.56±2.11, 4.63± 0.98(%), 고영양에서 10.67±2.00, 7.33±2.18(68.69%) 그리고 저영양에서 14.76±2.11, 10.94 ±1.91(74.11%)로 저영양에서 더 높게 나타났다. 본 연구 결과들로부터 일시적인 영양수준 조절을 이용해 체내수정란 생산 극대화 기술을 확립할 수 있고, 이러한 기술개발을 이용하 여 고능력 한우의 조기번식, 한우개량 및 농가소득증대에도 크게 기여할 것으로 사료된다.

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.

Poly(ADP-ribosyl)ation is post-translational modification of cellular proteins related to cell survival, cell death, cellular proliferation and epigenetic events. It has recently been shown to be important for pre-implantation development of mouse embryos. However, its function during early embryonic development of pig is not clear. This study investigated the importance of poly(ADP-ribosyl)ation during in vitro development of pig embryos produced by in vitro fertilization(IVF) or parthenogenetic activation (PA). Results showed that, chemical inhibition of PARP by 3-aminobenzamide (3-AB) did not influence the in vitro development of pig embryos up to morula stage (20±3.1 vs. 28.1±1.2%; p>0.05) but significanlty reduced the rate of blastocyst formation (5.2±2.1 vs. 20±3.1%; p<0.05) when compared to non-treated controls. Furthermore, culture of morula stage embryos in the pressence of 3-AB for 24h significantly reduced the rate of blastocyst formation (19.6± 4.6 vs. 41.4±5.3%; p<0.05) and expansion (4.7±3.0 vs. 28.1±6.1; p<0.05). The proportion of large-sized blastocyst (>200 μm) having higher blastocoel volume (15.3×106 μm3) was significantly reduced (p<0.05) in treatment group (32.2±7.8%) compared to non-treated control group (65.7±9.0%). TUNEL assay revealed that poly(ADP-ribosyl)ation-inhibited blastocyst had significantly increased indices of apoptosis than those of non-treated controls (10.88±0.02 vs. 2.71±0.01; p<0.05). These data suggest that Poly(ADP-ribosyl)ation may be important for blastocyst formation in pig embryo.

Autophagy is known to be involved in a variety of biological processes. However, relatively a little is known regarding oocyte maturation and preimplantation development in mammals. Thus, the current study was conducted to investigate the role of autophagy in oocyte maturation and subsequent preimplantation development in pigs. Porcine oocytes were matured in the presence or absence of 1 μM rapamycin, an autophagy inducing agent, fertilized in vitro, and cultured to blastocyst stage. From Western blotting analysis, we found that active form LC3 was detected during in vitro maturation (IVM) period, suggesting the possible role of autophagy in oocyte maturation. Interestingly, treatment of rapamycin during IVM significantly increased nuclear maturation compared to control group. Importantly, rapamycin-assisted IVM greatly improved monospermic fertilization and blastocyst development rates compared to control embryos. In addition, we also found that cell number and blastomere survival in blastocysts were markedly increased in rapamycin treatment group, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Collectively, these results strongly suggest that induction of autophagy may contribute to the completion of nuclear and cytoplasmic maturation of porcine oocytes.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes. However, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism (s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress inearly embryos. Collectively, these results suggest that early embryo genesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Pig embryonic stem cells (ESC) has been suggested to become important animal model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, the quality of cloned embryo and derivation rate of cloned blastocyst has been presented limits for derivation of cloned embryonic stem cell. In this study, we have tried to overcome these problems by aggregating porcine embryos. Zonafree reconstructed SCNT Embryos were cultured in micro-wells singularly (non-aggregated group) or as aggregates of three (aggregated groups) at the four cell stage. Embryo quality of the cloned embryos and attachment on feeder layer rate significantly increased in the aggregates. The aggregation of pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of pig cloned blastocyst and improvement in the percentage of attachment on the feeder layer of cloned embryos. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

Cathepsin B, a lysosomal cystein protease that plays an important role in the degradation of intracellular proteins in lysosomes, is detected in a wide variety of cells including bovine oocytes and embryos. Although the mode of action of cathepsin B is not fully understood, a strong relationship was observed between cathepsin B and apoptosis in many types of cells. Cathepsin B was found to induce the apoptotic pathway through activating initiator caspases rather than executioner caspases. Thus, the aim of this study was evaluated the effect of capthesin B inhibitor, E-64, on blastocyst developmental competence and subsequent preimplantation quality of the IVF and SCNT bovine embryos. After IVF and SCNT procedures, presumptive bovine embryos were cultured in CR1aa medium supplemented with E-64 for 24 h. Then, samples were additionally cultured in CR1aa medium without E-64 for 5 days. In our results, the frequency of blastocyst formation was higher when treated with E-64 compared with the control group (p<0.05). Furthermore, the blastocyst cell number was enhanced and apoptosis reduced (TUNELpositive nuclei number) by E-64 treatment in both IVF and SCNT bovine embryos (p<0.05). In the real-time quantitative RT-PCR, the expression of anti-apoptotic Bcl-xL gene was shown to be increased in the blastocyst stage, whereas expression of proapoptotic Bax was decreased. In conclusion, our results indicate that E-64 improves the developmental competence and embryonic qualities of bovine IVF and SCNT embryos by modulating cathepsin B induced apoptosis during the preimplantation stage.

Although evidences showed that histone deacetylation plays an important role in the mitotic and meiotic cell cycle, but the mechanisms are still unclear. Level of histone acetylation can be easily changed by deacetylase inhibitors (HDACi) i.e trichostatin A (TSA) and valporic acid. In this study, we determined whether the inhibition of histone deacetylation by TSA could affect porcine oocyte maturation and aging process. Our results showed that treated COCs with 100 nM TSA significantly increase the GVBD in each time group than 0, 5, 50 nM but no significantly different from that of higher concentration (200 nm or 300 nM). No significant differences on maturation, blastocyst development, MAPK pattern and expressions of apoptosis gene when treated oocytes with 100 nM TSA for the first 24h of IVM compared with control and 5, 50 nM TSA. However, in the oocytes treated with 200 nM and 300 nM TSA for first 24 h, MAPK significantly decreased and abnormal spindle were observed. But, in prolonged (64 h) of TSA treated group has no significantly different in control. Another data observed that after 24h TSA-treat to prolonged group were significantly decreased of MAPK activation and normal spindle than the other group. We concluded that TSA played a critical role in meiotic progression in porcine oocytes through the regulation of arrest GVBD, which prolonging the in vitro maturation time, but unaffected the subsequent pre-implantation embryo developmental potential and embryonic qualities. Moreover, the histone deacetylase inhibitor TSA may artificially control porcine oocyte maturation time and delay porcine oocyte aging process.

Superovulation, or ovarian stimulation is a commonly used ART for treatment of human infertility/subfertility. Recent studies suggest that superovulation unaffects methylated imprints acquisition in mouse oocytes during oogenesis, whereas disrupts DNA methylation maintenance in embryos during preimplantation development. However, the mechanisms of defects in methylation maintanence caused by superovulation remain largely unclear. We hypothesized that superovulation may disrupt the expression of DNA methyltransferases (Dnmts), the enzymes which catalyze DNA methylation acquisition and maintenance. The mice were subjected to superovulate with low (6 IU) and high (10 IU) dosage hormone. We examined the global DNA methylation levels in zygotes and DNA methylation of repeated sequences (IAP and Line 1) in blastocyst stage embryos. In addition, we investigated the expression of Dnmts (Dnmt3a, Dnmt3b, Dnmt3l and Dnmt1o) in ovulated oocytes and zygotes. Through staining with antibody 5mC and Di-H3K9 coupled with confocal microscopy, we found that global methylation profiles in zygotes derived from females after low or high dosage hormone treatment were not affected when compared to control counterpart. Moreover, methylation at IAP in blastocysts also was unaffected by superovulation, irrespective of hormone dosage. In contrast, methylation level at Line 1 decreased when the females were administered by high dosage hormone. Furthermore, expression of de novo DNA methyltransferase Dnmt3a, Dnmt3b, Dnmt3L, as well as maintenance Dnmt1o in MII oocytes and zygotes was not disrupted by superovulation. Given superovulation adversely affected methylation maintenance in blastocysts during preimplantation development but with normal expression of Dnmts in oocytes and zygotes, it is indicated that defects of embryonic methylation didn’t originate from abnormal expression of Dnmts.

It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte. In this report, we elucidated the molecular requirements for actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues have recently been shown in female oocyte cells. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) may be considered a marker of apoptosis. Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stage that suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulations of apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis-related genes and PARP family genes. In conclusion, Doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

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.

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).

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.

Doxorubicin, a widely used chemotherapeutic agent, were found rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis of oocyte, and a little known is actions of this drug in early embryos. Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, also plays the important role during the apoptosis of cell. The cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Cleaved PARP (cPARP) can be a marker of apoptosis.Doxorubicin inhibited the early embryo development, but the treatment could still reach the BL (blastocyst) stagethat suggested that involved in DNA synthesis and repaired progress. Herein, the higher expression of PARP family shown especially in 2, 4 cell stagy. There was evidence of expression of Caspase3 and Bcl2l1 during embryogenesis (2 cell, 4 cell, morula and BL stage), suggesting that modulationsof apoptosis-related genes and PARP were cause by DXR. Furthermore, the effect of doxorubicin on early embryo development was assessed different stage rates, and apoptosis index also conformed doxorubicin modulate embryo development by regulating apoptosis- related genes and PARP family genes. In conclusion, Doxorubicin blocked pre- implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by Parp.

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.

Autophagy is conserved response to starvation by which cells catabolize their components to create an internal supply of essential nutrients. Ceramide is known to induce autophagy in many cells through down-regulation of amino acid and glucose transporters. The mechanism of starvation induced-autophagy in mouse embryo remains unclear. In order to understand the mechanism by which starvation regulates autophagy, in this study, we investigated nutrient transporters expression and the effect of c2-ceramide on the in vitro development, apoptosis and autophagy via starvation in mouse embryo. Glucose transporters (Glut1 and Glut 3), high levels of transcript were expressed from 1 to 2 cells and gradually decreased through the morula and blastocyst (BL) stages. Amino acid transporters (LAT-1 and 4F2hc) gradually decreased from the zygote to the BL stage. Furthermore, the expression of nutrient transporters (Glut1, 3, LAT-1 and 4F2hc) were significantly reduced at the BL stage after ceramide treatment. Especially, mTOR expression after ceramide treatment of embryos was significantly higher than controls. Ceramide treated embryos exhibited significantly reduced developmental rates and total cell numbers, and increased apoptotic cell death at the BL stage. Consequently, we next evaluated the effect of ceramide treatment on mitochondrial number and morphology. There was a significant decrease in the average mtDNA copy number and the mitochondrial area in ceramide treated BL stage embryos. Both the expression of autophagy-related genes, Lc3, Gabarap, Atg4A and Atg4B, and the synthesis of LC3 were significantly induced at the BL stage. These results suggest that autophagy under starvation condition influences the in vitro development and apoptosis and autophagy, and may play a role in early mouse embryogenesis.

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.

전광판식 번식 기록관리 시스템은 판넬식 전광판 형태로 되어 있고, 우사의 벽에 설치하 도록 제작되어 있어, 재발정일, 이전(以前) 수정일, 분만예정일, 건유일 등과 같은 관리일 (日)을 알고 싶을 때나 그밖의 개체에 관한 번식정보를 알고자 할 때는 수시로 확인할 수 있음은 물론 분만, 인공수정 등과 같은 관리사항을 기록해야 할 일이 생겼을 때는, 기입할 칸에 <날짜입력>이라는 키를 누름으로써 오늘 날짜가 자동적으로 기록됨과 동시에 저장이 되도록 고안되어 있어 현장에서 사용하기에 매우 적합하도록 되어 있는 기록관리의 편의성 을 제공하기 위해 고안된 시스템이다. 전광판식 번식 기록관리 시스템을 현장에 적용하여 도태율, 번식장애로 인한 도태율, 공태기간 및 공태기간 분포를 조사한 결과, 도태율은 5.7%, 번식장애로 인한 도태율은 10.3% 낮아졌고, 공태기간은 32.2일 단축되었으며, 공태 기간 121일 이상의 비율이 낮아지는 것으로 나타났다. 젖소 농장에서 1일 3회 이상 관찰을 하는 것은 정보 및 경영 활동 등으로 인한 목장에 부재하는 시간이 증가함에 따라 현실적 으로 어려움이 따르고, 또한 온도상승, 사육환경(사육밀도, 우사바닥)의 변화, 고능력우의 증 가, 심야시간대의 발정개시, 미약발정우의 증가 등으로 발정발견이 매우 어려운 상황이다. 발정발견 보조 수단으로는 발정발견의 가장 중요한 지표가 되는 승가허용 원리를 이용하여 소의 미근부에 부착하는 형태의 일회용 또는 전자식 보조기가 많이 개발되어 보급되고 있 는데, 여기서 공시한 발정탐색기는 승가 및 승가허용 행동을 할 때, 발목에 부착되어 있는 센서가 수직적인 충격에 의해 위와 아래의 전극이 접지되어 신호가 발생되고, 그 신호가 전 광판으로 전송되어 발정 정보(발정행동 횟수 및 개시시간)를 표시하도록 고안된 것이다. 승 가나 승가허용의 발정행동에 의해 90% 정확도와 75%의 수태율을 나타내었고, 젖소 사육 농가 5개소에 적용한 결과, 적용 전에 비하여 적용 후에 발정발견율이 크게 향상되는 경향 을 나타내었다. 무인 발정탐색기를 이용하여 발정이 개시되는 시간대를 조사한 결과, 발정 발견이 곤란한 저녁 7시부터 다음날 아침 7시 사이에 59.1%가 발정이 개시되는 현상을 나 타내었고, 발정행동을 한 최초의 시간으로부터 12시간 이후에 인공수정을 실시하는 것이 높은 수태율을 나타내었으며, 발정행동 횟수의 분포를 조사한 결과 3회 이하의 비율이 40.9 %를 나타내었다.