Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with Triladyl®-egg yolk diluent and then was performed cryopreservation.There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

정자 생존성은 형광염색법을 실시하여 현미경검사, 세포현광분석, flow cytometry 등 여러 방법 으로 평가될 수 있다. Flow cytometry는 정자의 형태적, 기능적 특징의 여러 가지 항목을 짧은 시 간내에 수천에서 수만개의 정자를 검사할 수 있는 방법으로 기존의 eosin-nigrosin 염색법, HOST의 실험실적 검사나 형광현미경 검사에 비하여 시간 경과에 따른 정액 성상의 변화를 최소화 할 수 있으며 보다 객관적인 결과를 얻을 수 있다. 이 연구는 Flowcytometry를 사용하여 제주흑우의 동결 정액에서 정자의 생존성을 평가하는 2가지 형광염색법의 비교분석을 통해 정자 생존율과 이중 형 광 염색법 유효성을 알아보고자 하였다. 국립축산과학원 난지축산연구소에서 사육중인 제주흑우 2두를 인공질법을 이용하여 채정하였으 며, Triladyl-eggyolk 희석제 희석하여 동결하였다. LN2에 동결보존된 정액 스트로(n=12)를 융해하여 PBS를 이용하여 희석 후 2개로 분획하였으며, 각각 Calcein Am/PI(CAM/PI)와 6-CFDA/PI(CFDA/PI) 의 이중 형광염색을 실시하고 Flow ctyometry로 생존율을 비교 분석하였다. 분석결과 살아있는 정 자의 비율은 29.26±1.28(CAM+/PI-), 27.50±0.76(CFDA+/PI-), 약간의 생체막의 손상이 있으나 살아있 는 정자의 비율은 21.67±4.92(CAM+/PI+), 23.29±2.76(CFDA+/PI+), 생체막의 손상으로 죽은 정자의 비율은 48.44±4.18(CAM-/PI+), 48.61±2.71(CFDA-/PI+)이었으며 각각 형광염색법간의 유의적인 차이 는 없었다(SPSS v18.0, Paired t-test) 본 연구결과 Flow cytometry를 사용하여 정자의 생존율을 평가할 때 CAM과 CFDA의 차이는 존 재하지 않는 점을 알 수 있었으나 형광염색에 따른 세포의 분포를 비교했을 경우 CFDA가 CAM 보다 형광염색에 따른 정자세포 집단의 구분이 명확하였다. 따라서 정자의 생존성 평가시 CFDA 와 CAM은 모두 사용가능하지만, 정자세포의 집단의 구분에 따른 다른 검사를 실시할 경우 CFDA/PI 염색법이 유용하다고 판단된다.

정자 생존성은 정자기능평가에서 중요한 부분이며 Eosin-nigrosin 염색법, Hypo osmotic swelling test법, CFDA, SYBR-14, Hoechst-33342, Calcein AM 등의 형광염색법을 통해 평가될 수 있다. 또한 Flow ctyometer를 이용, 단시간에 형광 염색된 세포를 검사하여 생 존성뿐만 아니라 정자의 여러 기능적 특성을 평가하는 기술이 최근 이용되고 있다. 이 연 구는 Flowcytometry를 사용하여 제주흑우의 동결정액에서 정자의 생존성을 평가하는 2가 지 형광염색법 간의 비교분석을 통해 정자 생존율의 차이가 있는지 알아보고자 하였다. 국립축산과학원 난지축산연구소에서 사육중인 제주흑우 2두를 인공질법을 이용하여 채 정하였으며, 정자 최종농도가 2.0×107/ml가 되도록 Triladyl-eggyolk로 희석 후 4℃에 1시 간 30분간 평형과정을 수행하였다. 그 후 스트로 정액 충전기로 정액을 충전 후 간이 액 체질소 증기 동결법으로 동결하여 LN2에 동결보존하였다. 동결보존된 정액 스트로(n=12) 를 융해하여 PBS를 이용하여 5×105/ml 농도로 희석 후 2개로 분획하였으며, 각각 Calcein Am-PI(CAM/PI)와 6-CFDA-PI(CFDA/PI)의 이중 형광염색을 실시하고 37℃, 15분간 정치 후 Flow ctyometry로 생존율을 비교 분석하였다. 분석결과 살아있는 정자 의 비율은 29.26±1.28(CAM+/PI-), 27.50±0.76(CFDA+/PI-), 약간의 생체막의 손상이 있으나 살아있는 정자의 비율은 21.67±4.92(CAM+/PI+), 23.29±2.76(CFDA+/PI+), 생체막의 손 상으로 죽은 정자의 비율은 48.44±4.18(CAM-/PI+), 48.61±2.71(CFDA-/PI+)이었으며 각 각 형광염색법간의 유의적인 차이는 없었다(SPSS v18.0 통계프로그램 사용). 본 연구결과 Flow cytometry를 사용하여 정자의 생존율을 평가할 때 Calcein AM과 6-CFDA의 차이는 존재하지 않는 점을 알 수 있었으며, 정자의 생존성 평가에 CAM/PI 방법과 CFDA/PI 방법 중 하나를 선택하여 사용 가능하다고 판단된다.

최근 가축의 유전적 다양성 유지 및 식량 안보에 있어서 재래품종의 중요성은 점차 증대되고 있다. 제주흑우는 멸종위험에 처한 품종이며, 2013년 7월 문화재청에 의해 천연기념물 제546호로 지정되었다. 본 연구의 목적은 제주흑우(124두)의 유전적 다양성, 유연관계 및 유전적 구조의 평가이며, 그 비교 대상으로 한우(128두) 및 외래품종 홀스타인(73두)을 공시하였다. 분자유전학적 특성을 평가하기 위해 11개 초위성체 마커(BM1824, BM2113, ETH10, ETH225, ETH3, INRA23, SPS115, TGLA122, TGLA126, TGLA227, TGLA53)의 대립유전자형을 분석하였으며, 그 결과를 토대로 유전적 다양성 지수들을 산출하였다. 품종별 평균 기대이형접합도(HExp)는 0.605-0.738, 관측이형접합도(HOsb)는 0.667-0.747 그리고 다형정보지수(PIC)는 0.644-0.773의 범위를 보였다. 특히, 제주흑우의 유전적 다양성 지수는 가장 낮은 결과를 보였다. STRUCTURE를 이용한 군락 분석 결과 유전적으로 3개의 군락으로 구분되었으며, 주성성분분석(PCA) 결과 또한 3개의 군집으로 분류됨을 확인하였다. 따라서 본 연구의 결과는 제주흑우의 유전적 고유성 및 유전자원으로써 가치 판단을 위한 과학적 근거가 될 것으로 사료된다.

This study was carried out to examine a molecular marker system for parentage test in Jeju Black cattle (JBC). Based on the preliminarily studies, we finally selected for construction of a novel genetic marker system for molecular traceability, identity test, breed certification, and parentage test in JBC and its related industrial populations. The genetic marker system had eight MS markers, five indel markers, and two single nucleotide polymorphisms (SNPs; g.G299T and g.del310G) within MC1R gene which is critical to verify the breed specific genotypes for coat color of JBC differing from those of exotic black cattle breeds such as Holstein and Angus. The results showed lower level of a combined non-exclusion probability for second parent (NE-P2) of 4.1202×10-4 than those previously recommended by International Society of Animal Genetics (ISAG) of 5.000×10-4 for parentage, and a combined non-exclusion probability for sib identity (NE-SI) of 2.679×10-5. Parentage analysis has been successfully identified the JBC offspring in the indigenous population and cattle farms used the certified AI semens for production using the JBC-derived offspring for commercial beef. This combined molecular marker system will be helpful to supply genetic information for parentage test and traceability and to develop the molecular breeding system for improvement of animal productivity in JBC population.

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of 2×108/ml by doubling in every 30 minutes at 4℃ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at 37℃ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm (93.27±1.62%) than frozen-thawed sperm (73.34±3.27%). However, there were no significant differences between fresh and frozen-thawed dead cell rate (7.35±2.63 vs, 13.71±2.85). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different (72.86±2.83 vs, 81.47±2.48), similarly, the dead cell rates was similar (18.41±3.42% and 17.26±4.25). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY (; EG and ; 7% G) than 8% LDL (; EG and ;G). Treatment of 4% LDL + 5% EY-EG () has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG () among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

This study was carried out to investigate effective condition for producing somatic cell nuclear transfer (SCNT) embryos of Jeju native cattle. As donor cells for SCNT, ear skin cells from Jeju native cattle were used. In experiment 1, the effect of recipient oocyte sources on the development of Jeju native cattle SCNT embryos were examined. Fusion rate of recipient oocyte and donor cell was not different between the Hanwoo and Holstein recipient oocytes (86.0% vs 89.9%). The rate of embryos developing to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein recipient ones (28.2% vs 14.7%). Blastocysts derived from Hanwoo recipient oocytes contained higher numbers of total cells than those derived from Holstein ones ( vs ), although there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the sources of recipient oocytes. In experiment 2, the development of Jeju native cattle and Hanwoo SCNT embryos were compared. Hanwoo oocytes were used as the recipient oocytes. Fusion rate was not different between the Jeju native cattle and Hanwoo SCNT embryos (92.1% vs 92.9%). The blastocyst rate of SCNT embryos was significantly (p<0.05) lower in Jeju native cattle than in Hanwoo (16.9% vs 31.0%). Blastocysts derived from Jeju native cattle SCNT embryos contained smaller numbers of total cells than those derived from Hanwoo ones ( vs ), but there were no significant difference. The mean proportion of apoptotic cells in blastocyst was not different between the Jeju native cattle and Hanwoo SCNT embryos. The present study demonstrated that Hanwoo recipient oocytes were more effective in supporting production of Jeju native cattle SCNT embryos, although Jeju native cattle SCNT embryos showed reduced developmental capacity when compared to Hanwoo SCNT embryos.

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into LN2. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol (72.5±5.00%, 54.88±0.66% and 46.00±2.40%; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol (34.69±4.64% vs 46.00±2.40%; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: 55.81±2.94, 55.19±3.34 vs 47.94±3.48%; p<0.05 and membrane integrity: 44.94±3.51, 46.06±2.25 vs 40.38±1.03%; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN. The presented straws were examined the viability and motility after thawed at 37 water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 (≥ 2-cell) embryos were cultured in 0 (control), 1, 10 and 20 μM flavonoid for 6 days. In the results, in vitro development rate was the highest in 10 μM flavonoid group (57.1%) among treatment groups (control, 49.5%; 1 μM, 54.2%; 20 μM, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in 10 μM flavonoid group than other groups. We found that 10 μM flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in 10 μM flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, 10 μM flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in 10 μM flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

본 연구는 제주도의 흑우에서 다배란을 유기한 후 발정기 동안 혈중 호르몬의 농도 검사, 혈액 생화학 검사, 체내 수정란 회수율 등을 관찰하였다. FSH를 4일간 8회 50 mg씩 주사하여 다배란을 유기하였다. 성호르몬의 측정은 radioimmunoassay (RIA)법으로 측정하였으며 혈액 생화학치는 자동혈청 분석기로 측정하였다. 인공 수정 후 7일에 수정란을 비외과적 방법으로 회수하였다. 본 연구의 결과는 다음과 같았다. 1. 혈중 호르몬의 농도를 측

본 연구는 년(5년간) 제주도에서 사육중인 제주 한우와 흑우를 체내 수정란의 생산과 수정란 이식 기술을 통하여 조기 증식하고자 실시하였다. 한우 고등 등록우 286두와 흑우 경산우 69두(총 355두)에 대하여 발정 주기에 관계없이 CIDR를 질내에 삽입 후 7일째부터 성선 자극 호르몬() 400 mg을 50 mg씩 균등하게 나누어 4일간 12시간 간격으로 근육주사하였다. 투여 6회째에 CIDR를 제거하였으며, 동시에 25mg을 근육 주사하여 과배란을