In mammals, the meiosis division in testes produces equal numbers of two different types of gametes: X chromosome-bearing sperm (X-spermatozoa) and Y chromosomebearing sperm (Y-spermatozoa), which have equal potential to fertilize the oocytes. Therefore, the expected 1: 1 sex ratio is observed. However, under some conditions like endocrine disruptors (EDs) exposure the sex ratio is deviated than the expected with more males or more females. And recently many hypotheses have been postulated to explain the mechanism of sex ratio deviation; however none of them introduced a proven experimental explanation. To solve this enigma, we hypothesized that the differences between X- and Y-spermatozoa survivability under specific conditions due to differences in their chromosome contents are the key leading to the sex ratio alteration. To examine our hypothesis, we combined two techniques; first, hypo-osmotic swelling (HOS) test that was applied to test viability of spermatozoa and second, fluorescence in situ hybridization that was applied on HOS-treated spermatozoa to define sex chromosome composition. In the present study, human spermatozoa were incubated with a group of EDs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, the viability of spermatozoa and their sex chromosome contents were evaluated simultaneously. Among seven chemicals studied only four chemicals (Atraz, DBCP, TCDD, and Diaz) significantly decreased Y-sperm viability when compared to those of X-spermatozoa in the same treatment group and viability of Y-spermatozoa when compared to those in the negative and positive (DMSO) control groups (p<0.05). Also, in these four treatment groups the sex ratio of live sperm population was significantly lowered compared to the control groups (p<0.05). Otherwise, Gen, BPA, and NP did not show any significant effect on viability of Yspermatozoa or decreasing sex ratio in live sperm population as compared to the control groups. It has been proven that TCDD, DBCP, and the pesticides decrease the sex ratio, but the same effect was not observed in case of Gen, BPA, and NP. From the present findings, there is no doubt that the EDs may alter sex ratio via decreasing Y-spermatozoa viability.

Phosphorylation of proteins is a post-translational modification process which plays a significant role in a wide range of cellular processes. Addition or removal of phosphate groups result in conformational changes in proteins leading either to their activation or inactivation. Tyrosine phosphorylation of protein is associated with sperm function in several mammalian species. The control of this process may via the changes in cyclic adenosine monophosphate (cAMP); the changes in cAMP levels that occur in the spermatozoa regulate protein kinase A (PKA) activity which, in turn, leads to the tyrosine phosphorylation of protein substrates by either the activation of sperm tyrosine kinases and/or the inhibition of phosphoprotein phosphatases. Cyclic nucleotides, in particular, cAMP, are important regulators of various maturation events in sperm including capacitation and motility. Interestingly, some environmental chemicals (ECs) may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems. Otherwise, because the mature spermatozoa are transcriptionally inactive, therefore the study of sperm proteins phosphorylation may permit more information about the agents and conditions affects on sperm function. In the present study, to examine the effect of ECs on human sperm function, human spermatozoa were incubated with a group of ECs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo- pdioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, western blot analysis was carried out using extracted sperm proteins. Antiphosphorylation antibody (pY20) was used to determine sperm tyrosine phosphorylation after EDs treatment. The pY20 antibody labeled three common bands of approximately 90, 110, and 150 KDa. There were no significant differences between negative and positive control groups in regard to the tyrosine phosphorylated proteins except at the band with molecular weight 110 KDa. However, except Diaz treatment group, the other treatment groups showed decreasing (TCDD, Gen, NP, BPA, and DBCP) or increasing (Atraz) in the tyrosine phosphorylated proteins at least in one band from the three common bands studied. Therefore, it sug-gests that ECs effectively alters human sperm function and this effect may detect via their effect on tyrosine phosphorylation pattern.

Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300 X g and 700 X g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

The prediction of male fertility is of paramount importance for breeding animal herds when artificial insemination is applied. While the male fertility assays provide valuable quantitative data, they yield limited information concerning the functional competence of the spermatozoa. The objective of this study was to standardize a method for predicting in vivo fertility in bulls using the capacitation status that was assessed by chlortetracycline (CTC) staining. To optimize the capacitation process, sperm were treated with various concentrations of heparin (0, 10, 20, 50, and 100 μg/mL) and incubated for 10, 20, and 30 min each at 39℃ in 5% CO2. We found that maximum capacitation condition obtained from 10 μg /mL heparin treated sperm cells for 20 min (p<0.05). Optimized methods were used to determine the fertility of 17 batches of frozen bull semen representing a wide range of field fertility levels as indicated by non-return rates (NRR) (35.29% 93.18%). There was no significant correlation between NRR and the percentage of capacitated spermatozoa (B type) and non-capacitated spermatozoa (F type). However, acrosome reacted spermatozoa (AR type) was significantly correlated with NRR (p<0.01). To determine the normal range for the AR type, lower limits of the AR (%) were established as 23% for low fertility (NRR < 75%) using receiver operating characteristic curve. The overall accuracy of the assay was 88.24% for low fertility, sensitivity and specificity were 81.82 and 100%, respectively. These results indicate that capacitation status as measure by CTC staining is a useful predictor of male fertility. Therefore, low and high fertility bulls can be identified primarily by the functional capacitation status.

This study was focused on improvement of milk production in Mongolia dairy industry by artificial insemination (AI) technology, of which was supported from ODA project of KOICA in Republic of Korea. The study was started from January 2009 to present and 3rd years in this year. So, all data, especially synchronization and pregnancy of dairy cows (Holstein) will be summarized in final result in this year. For synchronization, total 81 dairy cows selected from 4 private farms that were 38, 30, 8 and 5 in Undarmal milk, Onjin (Enkhbayer), Jargalant, and BRM School, respectively. All the dairy cows were injected intramuscular (IM) of 5 ml PGF2α in the vulva and detected estrus 2 to 3 days after PGF2α injection. Total 78 out of 81 dairy cows (96.3%) were detected estrus by only 1 time injection of PGF2α. The dairy cows that were induced estrus, inseminated with 0.5 ml dairy frozen semen by conventional AI techniques. The pregnancy diagnosis of the AI dairy cows was detected by uterus palpation after 60 days of insemination. Total 75 from 78 inseminated dairy cows (90.1%) were diagnosis pregnant. The estrus induction and pregnant rate were very effective using PGF2α injection and conventional AI techniques in Mongolia dairy cow. The results indicated that AI after estrus induction in Mongolia dairy cows could be applied to dairy breeding technology to improve the breeding efficiency and milk production.

최근 재래가축 유전자원의 중요성이 부각되면서 그 중의 칡소의 유전형 확립이 대두 되고 있다. 칡소는 호반모를 가진 종모우의 정액을 이용하여 수정하여도 호반모가 아닌 일반 한우의 모색과 비슷한 양상으로 출현하는 개체들이 있다. 이는 칡소 모색 관련 유 전자들의 발현이 고정되지 않았다는 가능성을 제시한다. 소의 모색관련 유전자 중에서 melanocortin 1 receptor(MC1R) 유전자의 결실은 한우의 모색 및 한우고기의 판별에 이 용되어 왔다. 본 연구는 PCR-RFLP 기법으로 melanocortin 1 receptor(MC1R) 좌위의 유 전자형 검출을 통해 칡소와 한우의 유전자형 빈도와 염기 서열상의 차이를 비교 분석함 으로써, 칡소의 모색 발현에 관련된 MC1R 유전자를 이용하여 호반모 발현 비율과 유전 양상을 밝히기 위하여 수행되었다. 본 연구는 전라북도 축산위생연구소 축산시험장에서 사육 중인 칡소로 부계나 모계를 알고 있거나 가계가 형성된 칡소로 부터 혈액을 채취하여, Genomic DNA를 추출하였다. MC1R 특이 유전자 마커를 이용하기 위해 GenBank(S71017)에 등록된 염기 서열을 참고 하여 Primer를 설계하였다. MC1R 유전자의 증폭을 위한 PCR 반응 조건은 95℃에서 10 분간 정치 후 95℃ 30초, 60℃ 30초, 72℃ 1분 총 35 cycle을 수행한 뒤 72℃에서 5분 간 반응시켜 PCR을 완료하였다. 증폭된 MC1R 유전자 739 bp산물을 MspI으로 3시간 처 리 후 전기 영동하여 DNA 절편을 확인하였다. PCR 증폭 산물을 MspI 처리한 결과, 한우의 절편 양상은 2개의 절편(534 bp, 207 bp) 으로 대부분 나타났으며, 칡소는 4개의 절편(535 bp, 328 bp, 207 bp, 174 bp) 양상이 3 개의 절편(328 bp, 207 bp, 174 bp) 양상보다 다소 많은 경향을 보였다. 이 연구에서 얻 어진 한우와 칡소의 유전자 절편 양상을 비교 분석하여 호반모 발현비율을 증가시키는 교배체계의 확립에 이용될 수 있을 것으로 기대된다.

분만 후 최적 수정시기를 탐색하기 위한 초기의 연구에서는 분만 후 경과일 수에 따라 분만 후 60일 이내, 61 90일 및 90일 이상으로 구분하여 시험이 수행되었는데 처리별 첫수정 수태율이 각각 48%, 70% 및 76%였고, 수태당 수정횟수가 2.09회, 1.55회 및 1.54회였다고 보고하였으며(George, 1955), 분만간격이 360 374일인 젖소의 첫수정 수 태율에 있어서 분만 후 0 45일 38%, 46 60일 47%, 61 90일 55% 91 120일 59% 및 121 180일 59%로 분만 후 일수가 경과됨에 따라 수태율이 증가되는 양상을 나타내었다 고 보고하였다(Bozworth 등, 1972). 그리고 분만 후 50일 및 80일에 발정이 발견된 경우 의 수태일수는 각각 88일 및 121일이었고, 수태당수정횟수는 각각 1.50회 및 1.96회였다 고 보고하였다(Schneider 등, 1981). 또한, 분만 후 50 75일, 76 100일 및 100일 이상 에서 인공수정 시 임신율이 각각 36%, 47% 및 43%로 76 100일이 가장 높은 것으로 보고되었다(Pursley 등, 1998). 분만 후 비유단계에 따른 발정발현율, 수태율 등이 연구자 들에 따라 다소 의견을 달리하고 있고, 특히 산유능력(평균능력 및 고능력)에 따른 조사 는 이루어지지 않고 있다. 따라서 본 연구에서는 비유단계 및 산유능력에 따른 발정발현 율, 수태율을 조사하고, 차기의 번식에 좋지 않은 영향을 주지 않는 적정 수정시기를 구 명하기 위하여 수행하였다. 분만 후 기간에 따라, 분만 후 30 59일(30일구), 60 89일(60 일구), 90 110일(90일구) 및 111일 이후(111일구)로 구분하여 조사하였다. 분만 후 발정 발현율은 30일구가 16.7%(17/102), 60일구가 28.4%(29/102), 90일구가 20.6%(21/102) 및 111일구가 34.3%(35/102)였고, 분만 후 수태율은 30일구가 52.9%(9/17), 60일구가 51.7 %(15/29), 90일구가 47.6%(10/21) 및 111일구가 48.6%(17/35)였다. 산유능력별로 보면, 발정발현율에 있어서 30일구의 경우 평균능력우 및 고능력우가 각각 25.0% 및 16.7%, 60일구의 경우 각각 40.6% 및 20.8%, 90일구의 경우 25.0% 및 12.5%, 111일구의 경우 9.4% 및 50.0%였고, 수태율에 있어서 30일구의 경우 평균능력우 및 고능력우가 각각 87.5% 및 25.0%, 60일구의 경우 각각 61.5% 및 0%, 90일구의 경우 75.0% 및 33.3%, 111일구의 경우 66.7% 및 50.0%였다.

지속적인 젖소의 능력개량과 사양기술의 발전에 힘입어 우리나라 젖소의 우유생산 능 력이 점차 향상되는 추세에 있다. 2000년에 8,086kg이었던 검정 유량이 2010년 9,638kg 으로 증가되었고, 검정농가의 비율도 2000년에 25.8%에서 2010년 55.2%로 사업에 참여 하는 농가의 비율이 빠르게 증가되고 있다. 또한, 농가별 사육규모도 점차 증가되어 2000년에 호당 40.7두에서 2010년 67.7두로 증가되는 추세이다. 근래에 기후변화, 경영 의 복잡화, 우유 생산량과 호당 사육두수의 증가 등으로 농가에서 젖소의 번식관리에 상 당한 어려움을 겪고 있다. 번식관리의 어려움으로 번식 성적이 저하되는 것은 물론 도태 산차가 단축됨으로써 농가의 경제적 손실을 크게 하고 있다. 현재 우리나라 젖소의 평균 보유산차는 2.5산, 평균 도태산차는 2.9산으로 우유생산 능력이 최대로 발휘하기 전에 도 태되고 있는 것이 현실이다. 따라서 본 연구에서는 젖소의 도태 유형을 분석 및 비교하 고, 농가별 평균능력우의 보유 수준에 따른 경제수명 연장 가능성을 검토하기 위하여 수 행하였다. 평균능력우의 보유 비율에 따라 80% 이상(80% 이상구)인 농가 15호와 80% 이하(80% 이하구)인 농가 14호로 구분하여 분석하였고, 평균능력우의 비율이 80% 이상 인 농가는 896두, 이하구의 농가는 785두 총 1,681두를 대상으로 조사하였다. 평균 보유 산차는 80% 이상구의 경우 2.7산, 이하구의 경우 2.4산으로 평균 2.55산이었고, 평균 도 태산차는 80% 이상구의 경우 2.9±0.2산, 이하구의 경우 3.0±0.2산이었으며, 평균 도태율 은 80%이상구가 17.7%. 이하구가 17.0%였다. 주요 도태 유형을 보면, 번식장애가 34.5 % (101/293)로 가장 높았고, 다음으로 질병이 17.1%(50/293)로 나타났다. 번식장애의 경 우 80%이상구 및 이하구가 각각 33.3%(53/159) 및 35.8%(101/293)였고, 질병의 경우 각 각 20.8%(33/159) 및 12.7%(50/293)였으며, 유방관련 도태의 경우 각각 12.6%(20/159) 및 15.7%(21/134)였다. 주요 번식장애 유형별로 보면, 저수태의 경우 80%이상구 및 이하 구가 각각 41.5%(22/53) 및 72.8%(35/48)였고, 무발정의 경우 각각 39.6%(21/53) 및 16.7% (8/48)였다. 질병의 유형별로 보면, 소화장애가 30.0%(15/50)로 가장 높았고, 대사 장애가 26.0%(13/50)로 그 다음을 차지하였다. 유방관련 도태에 있어서는 유방염으로 인 한 경우가 85.4%(35/41)로 가장 높게 나타났다. 기립불능 유형으로 보면, 원인불명이 47.7 %(21/ 44), 운동기관 장애가 20.4%(9/44) 및 사고가 15.9%(7/44)였다. 주요 도태원인별 산차를 보면, 번식장애가 2.7산, 질병이 3.0산 및 유방관련 도태가 3.8산이었다.

Ovulation resembles a tissue remodeling process such as a blood coagulation. The present study was aimed to examine the involvement of tissue factor, a primary factor for extrinsic coagulation pathway, in the ovulation. Northern blot analysis revealed that mRNA levels of tissue factor and tissue factor pathway inhibitor 2 (TFPI-2) in the ovary were stimulated by human chorionic gonadotropin (hCG) treatment in surperovulation model, of immature rats. Real-time PCR analysis demonstrated that the expression of tissue factor and TFPI-2 was stimulated in granulosa and theca cells of preovulatory follicles, respectively. The induction of tissue factor mRNA was blocked by the progesterone receptor antagonist RU486. Tissue factor protein was not detected in the ovary by Western blot and immunohistochemical analysis due to the lack of a specific antibody. Interestingly, the levels of tissue factor and TFPI-2 mRNA were increased in the ovarian cells of rats induced ovarian hyperstimulation syndrome (OHSS) and in granulosa cells of OHSS patients undergiong in vitro fertilization. The present findings indicate the stimulation of tissue factor system during ovulation, and in OHSS patients, implicating the possible involvement of tissue factor system in OHSS.

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt- 1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5’-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in ‒ 640 bp to ‒ 30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the in vivo or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the in vivo was increased up to 84.38% in the SCNT embryo, moreover, de novo methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the in vivo or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

An understanding of oocyte gene expression is a necessary for the study of biological development. Recently, Oocyte has been used in many techniques such as somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) and embryonic stem cell derivation. However, the molecular mechanism underlying porcine oocyte is still unclear. In this study, we present the description of the porcine oocyte proteome. Proteins within the isoelectric point ranges of 3.0 to 10.0 were analyzed separately using 2‐dimensional electrophoresis (2‐DE). About 450 spots were detected in 2‐ D gel of oocytes, stained with Coomassie blue. Subsequent excision of 227 spots from gels and MALDI‐TOF MS analysis allowed the identification of 85 proteins. Our results indicated the composite profiles of proteins in the porcine oocyte. Tubulin beta chain and meiosis‐specific nuclear structural protein 1 antibody was used to confirm those antibody expression levels in immature, mature and parthenogenetic embryo. Western blot analysis showed that expressions of those proteins increased during mature and parthenogenetic embryo. These protein profiles will make available important guides for the study of oocyte function and assist in functional analysis of the proteins.

Covalent modifications of histone tails have fundamental roles in chromatin structure and transcriptional activity of a target locus. One of such modifications, Methylation at Lysine 9 of histone H3 (H3-K9) causes several epigenetic phenomena including heterochromatin formation, transcriptional regulation and DNA methylation. Setdb1, H3-K9 specific histone methyltransferase, functions in gene silencing, heterochromatin formation and essential role for early development. Here, we demonstrate that Setdb1 associates with promyelocytic leukemia (Pml) protein from the early stage of mouse development and is a constitutive member of PML nuclear bodies (PML-NBs) that have been linked to many cellular processes such as apoptosis, DNA damage responses, and transcriptional regulation. Immunostaining of mouse blastocyst showed that Setdb1 and Pml signals were scattered in nucleus as a few speckles and microinjected Pmlmyc signals colocalize with Setdb1 signals. This colocalization was observed in mEF and the punctate signals of Setdb1 were observed to be present in every nucleus of mEFs and dividing cells with condensed chromosomes. Arsenic treatment, which induces Pml degradation, also caused Setdb1 signals to disappear. Setdb1 knockdown resulted in disassembly of PML-NBs and immunoprecipitation results demonstrated physical interactions between Setdb1 and Pml. These data suggest that Setdb1 was associated in PML-NB and Setdb1 has important function in maintenance of PML-NB structure.

The objective of this study was to evaluate the effect of Tea-N-tris medium on the sperm viability and acrosomal morphology for semen of normal and miniaure pig by type of freezing extender. The present study was to determine of Tea-N-tris (0.02 g/ml) effect to freezing extender LEY(Lactose 11% + Egg yolk 20%) and FGE(Fructose 3%+Glucose 7%+Egg yolk 20%) for the spermatozoa viability, acrosomal morphology and DNA fragmented analysis from normal and miniature pig semen, were evaluated freezing extender TFGE, TLE and LEY during thawing at 37℃ for 45 sec and 75℃ for 5 sec, respectively. Interestingly, the result that sperm after addition of Tea-N-tris extender(TFGE, TLE) during 15 4℃ cooling significantly increased the viability(p<0.05), as compared to than of sperm cooling in LEY extender, but lower the percentage of AR(acrosome reacted spermatozoa) pattern than LEY extender. The sperm viability and AR pattern after freezing was appeared like sperm cooling method pattern. And treatment spermatozoa during freezing after addition of Tea-N-tris extender significantly (p<0.05) increased the viability and AR to miniature pig sperm, than normal pig sperm, but most highly percentage of viability and AR pattern to normal pig sperm during freezing in LEY extender. Chromosomal DNA fragmentation increased from LEY extender to sperm of normal and miniature pig, but decreased from the Tea-N-tris extender. Therefore, suggest that Tea-N-tris freezing extender method for freezing of miniature pig sperm is required for increasing viability. This Study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

한우(韓牛, Korean Cattle, Bos taurus coreanae)는 모색에 따라 황소, 칡소, 흑소로 나 누어지고 있다. 칡소는 현재 1,700여두가 전국에 사육되고 있는 것으로 추정하고 있다. 그러나 종모우로 활용 가능한 수소의 숫자가 매우 적어 근친의 위험도가 높다. 본 연구 에서는 우량 칡소를 선발하기 위하여 성장단계별 체형을 측정하고, 12개월 및 30개월 령 에 신선정액과 동결융해 정액의 활력을 비교하였다. 칡소의 발육단계별 체형은 체고, 흉위, 십자부고, 체장, 흉심, 흉폭, 고장, 요각폭, 곤폭, 좌골폭을 각각 6개월, 12개월, 18개월, 24개월 및 36개월 이상으로 구분하여 측정하였다. 정액 채취는 인공질을 이용하여 채취하였고, 정액동결용 완충액은 Triladyl를 이용하였다. 칡소 수소의 성장단계별 체형을 조사한 결과, 12개월령 흉위, 체고, 십자부고, 체장, 흉 심, 흉폭, 고장, 요각폭, 곤폭 및 좌골폭이 각각 평균 156.0 cm, 113.2 cm, 118.6 cm, 122.6 cm, 59.5 cm, 31.7 cm, 43.0 cm, 34.7 cm, 36.6 cm 및 19.8 cm였고, 30개월령에 는 각각 평균 214.8 cm, 140.5 cm, 140.5 cm, 179.3 cm, 83.2 cm, 48.6 cm, 62.3 cm, 53.9 cm, 51.0 cm 및 34.7 cm로 조사되었다. 칡소 육성우의 모색을 조사한 결과, 전체 호반 무늬를 가진 개체는 9.6%, 부분 호반 무늬는 59.0%, 호반 무늬 없이 갈색인 개체는 20.5% 그리고 흑색인 개체는 10.8%였다. 15개월령 칡소와 30개월령 칡소의 신선 및 동 결융해 정액의 활력을 조사한 결과, 15개월령 수소의 정액량이 평균 2.3ml로서, 30개월 수소의 정액량의 평균 5.0ml로서 유의차가 인정되었다(p<0.05). 채정 직후의 신선정자의 생존율은 15개월 및 30개월 수소가 각각 평균 93.7% 및 88.3%, 운동성은 각각 97.0% 및 88.3%로서 운동성은 15개월령이 유의하게 높았다(p<0.05). 한편 동결융해 정자의 생 존율은 각각 평균 56.0% 및 58.0%였고, 운동성은 각각 평균 64.0% 및 70.7%로서 차이 가 없었다. 본 연구를 통하여 칡소의 동결정액 생산을 위한 체형이 우수한 개체의 선발이 가능하 였으나, 대량 증식을 위한 추가적인 연구가 필요할 것으로 생각된다.

돼지 정액 채취과정은 무균적으로 진행되기가 어려우므로 채취된 정액의 세균오염 (bacteriospermia)은 일반적인 것으로 알려져 있다. 돼지 정액에서 주로 분리되는 오염 세 균은 그람음성균이고, 장내세균과(Enterobacteriaceae)에 속하는 것으로 조사되고 있다. 정액 내 세균오염은 오염 정도에 따라 정액의 품질과 수명에 부정적인 영향을 미치는 것 으로 보고되고 있다. 본 실험에서는 돼지 정액에서 순수분리한 대장균을 실험적으로 농 도별로 오염시켜 돼지 정액의 활력, 생존율 및 pH에 대한 대장균 오염의 영향을 확인코 자 하였다. 정자 농도와 세균오염 정도를 조절하기 위하여 항생제가 첨가되지 않은 시판 돼지 정 액 희석제(Seminark Pro)를 이용하였고 돼지 정자수 대비 대장균수가 4,000 : 1(T1), 400 : 1(T2), 40 : 1(T3), 4 : 1(T4)이 되도록 액상정액 시료를 제조하였다. 모든 시료는 17℃ 저온배양기에 보존하면서 유세포분석기(flow cytometer)를 이용하여 0일차, 1일차, 3일차 및 5일차에 각 시료별 정자 활력(미토콘드리아 함량 측정)과 생존율(Live/DeadⓇ 염색)을 측정하였고, 산도측정기(pH meter)로 정액 pH를 측정하였다. 정자의 활력은 T3, T4에서 0일차(17℃, 4시간 배양)부터 대조군(C)에 비하여 유의성 있 게 감소하였고, 모든 시료에서 3일차 이후부터 활력이 감소(p<0.05)하였다. 정자 생존율 의 경우에도 T3, T4에서 0일차부터 C에 비하여 유의성 있게 감소하였고, 모든 시료에서 3일차부터 생존율이 감소(p<0.05)하였다. 보존일 경과에 따른 정액의 pH 변화의 경우, C, T1 및 T2에서는 0일차에 pH 7.00 정도에서 5일차(p<0.05)에 pH 7.10 이상으로 높아진 반면, T3에서는 3일차(p<0.05)부터 pH 6.90 이하로 낮아져 5일차에는 pH 6.86이었고, T4 에서는 1일차(p<0.05)부터 pH 6.86 이하로 낮아져 3일차(p<0.05)와 5일차(p<0.05)에는 pH 6.65 이하로 낮아졌다. 본 결과로부터 돼지 정자수 대비 대장균수가 40 : 1(20×106 sperm cells/ml : 5×105 cfu/ ml) 이상으로 오염되었을 경우 오염 당일부터 5일차까지 대조군에 비하여 정자의 활력과 생존율이 유의성 있게 감소되었으며 40 : 1의 경우는 3일차부터, 4 : 1의 경우는 1일차부 터 정자의 사멸 혹은 세균오염에 의한 산패로 정액의 pH가 낮아지는 경향을 확인할 수 있었다. 이는 정액 내 세균오염이 정액의 품질과 수명에 부정적인 영향을 미친다는 보고 와 일치하며 위생적인 정액 채취를 위한 노력과 고품질 정액 공급을 위한 정기적인 모니 터링 검사 및 보존액 내 유효한 항생제 첨가가 중요할 것으로 사료된다.

Bacterial contamination reduces the semen quality, semen preservation, and cause of disease spread as well. Sperm fertility is essential factor of reproductive performance in swine. Sperm fertility is affected by semen quality such as sperm motility, abnormality, morphology, and rate of bacterial contamination. This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22 24 hr incubation from counting agar plate in which sperm dilute to 10 106 in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern A), characteristic of uncapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern C), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 (77.24±6.47, p<0.001) and 7 days (77.24±6.47, p< 0.001) after preservation compared to 1 (15.71±7.18) and 3 days(18.39±7.22) after preservation, respectively. Sperm viability was significantly lower (53.25±35.03, p<0.0001) at 7 days after preservation. Mohological abnormality of sperm was lower (p<0.001) at 1 (15.71±7.18) and 3 (18.39±7.22) days compared to (5 21.84±7.91) and 7 (22.59± 9.93) days after preservation. Acrosomal integrity and capacitation rate (pattern A) were significantly lower (p<0.001) from 5 days after preservation.

The event that occur in sperm during chemotaxis are only partly known. As a essential step of fertilization, sperm cells should undergo capacitation process inside female genital tracts. To understand the molecular event of calcium signals on sperm cells, Fluo 4 loaded spermatozoa was treated with follicular fluid. The motility of sperm was reduced by follicular fluids. Simultaneously, level of calcium in head and tail was also reduced for 5 10 second. The inhibition of sperm motility was believed as a reversible event, so the follicular fluid in graffiaan follicles in vivo could act as a selector on active spermatozoa that recover motility and calcium signals during ovulation. This suggested that the normal levels of calcium in sperm was also critical for active state of sperm cells and the follicular fluids during ovulation could inhibit the motility of sperm cell via calcium signaling.

Cell transplantation therapy using adult stem cells has recently been identified as a potential treatment for spinal cord injury (SCI). But, recovery after traumatic SCI is very limited. As dogs are physiologically much more similar to human compared with other traditional mammalian models in disease presentation and clinical responses, a number of researches demonstrated canis familiaris is a suitable model for human diseases. This study investigated the effect of transplantation of canine Mesenchymal Stem Cells (cMSC) and neural-induced cMSC (nMSC) to understand how these cells improve neurological function in canine SCI model. The differentiation of cMSC into neural precursor cells was induced in dulbecco’s modified eagle’s medium supplemented with N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. SCI was induced between T1 and T2 by surgical hemi-section in adult dogs, and then assigned to two groups according to the applied cell types (cMSC vs nMSC). Pelleted cMSC or nMSC were transplanted directly into the injured site after SCI, respectively. Analysis of motor function after transplantation was evaluated by modified Olby score. Magnetic resonance imaging (MRI), histological and immunohistichemical analysis were also performed. Functional recovery in group of cMSC was increasing gradually after transplantation and was higher than nMSC. In MRI, we could not confirm any difference between the cMSC and nMSC experimental groups. Immunohistochemically, beta3-tubuline and nestin were observed in injury site of two experimental groups with the expression level close to non-injured groups. Transplantation of mesenchymal stem cells could promote neuronal reconstruction and repair motor function in SCI. These showed mesenchymal stem cells could be a great candidate as a therapeutic tools in degeneration disease, and dogs could be used to explore human regenerative medicine as a promising animal model. This research was supported by iPET (Grants 110056032CG000), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.