The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Semen analysis is the most commonly used procedure to evaluate male fertility potential. This study was to evaluate the quality of 10 JBC bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A∼J grade]. The freezing medium (20% egg yolk plus 20% triladyl) was added in semen sample to a final concentration of 100×106 sperm/ml. For sperm cooling, diluted semen was filled in 0.5 ml plastic straws and then kept in refrigerator at 4°C for 2 h. They were placed in 7 cm over liquid nitrogen (LN2) vapor for 10 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility, vitality and morphology in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea), eosin-nigrosin stain and diff-quik kit. There was no difference in the motility of the fresh groups (87.4 ~ 100%), while it was difference in the frozen-thawed groups (42.8 ~ 98.6%) (p<0.05). The best motility was shown in JBC-B (100/fresh and 98.6%/frozen-thawed). There was significant difference in the vitality of the fresh group (19.8 ~ 59.2%) and frozen-thawed group (21.2 ~ 49.8%)(p<0.05). The highest vitality was also shown in JBC-B (59.2/fresh and 49.8%/frozen-thawed). Morphologically, in fresh semen the highest normal ratio was indicated in JBC-E (90.9%) and in frozen-thawed group the highest was in JBC-C (90.2%). These results demonstrated that the analysis including motility, vitality and morphology of fresh or frozen-thawed semen is valuable to select the high quality sperm using for reproduction.

Purpose The Jeju black cattle are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. The Jeju black cattle are rare breed and designated as national natural monuments in 2013. It is necessary to improve reproductive techniques for the preservation and proliferation of Jeju black cattle. Methionine acts as a precursor amino acid for glutathione in protection of cells from oxidative damage, and plays a vital role in detoxification. Low sperm motility causes infertility because when sperm do not have progressive motility, it was unable to reach the ovum. The purpose on this study was to investigate whether intake of L-methionine improves sperm motility and fertility. Materials and Methods 6 Jeju black cattles over 10 years of age are raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA. 6 Jeju black cattles was fed with L-methionine with 10g/day during 6 weeks. Semen of Jeju black cattle were collected by artificial vaginal technique. Collected sperm was diluted with AndroMed® extender and cryopreserved in 0.5 ml French straws. Fresh and freeze-thawed sperm viability and motility were evaluated by CASA. Results The viability and progressive motility of fresh spermatozoa(live spermatozoa means: from 74.41% to 80.22%, progressive motility means: from 80.60% to 95.40%) and freeze-thawed spermatozoa(live spermatozoa means: from 51.26% to 62.05%, progressive motility: from 29.68% to 45.44%) incresed after L-methionine ingestion. Conclusion The intake of L-methionine in genetically valuable cattle over 10 years of age seems to be useful for improving sperm function.

The national natural monument of Korea, Jeju Black Cattle (JBC), it is a native species with unique blood line. This cattle breed needs mass production and industrialization to further improve and preserve their characteristics. This study was to examine whether there were differences in in vitro developmental rates according to body weight (<300, 300 ~ 350, 350 ~ 400 and >400 kg) and grade (1++, 1+, 1, 2 and 3), and oocyte donors or non-donors. As a method of IVM, groups of ten cumulus oocyte complexes (COCs) were cultured in 50 μl droplets of maturation medium (TCM199 supplemented with 10% FBS, 0.2 mM sodium pyruvate, 1 μg/ml follicle-stimulating hormone, 1 μg/ml estradiol-17β) under mineral oil at 38.8℃ in an incubator with a 5% CO2 atmosphere for 22 to 24 h. For IVF, 44 ul IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 μl heparin and 2 μl PHE (20 μM peicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. For IVC, after 44±2 h of incubation, cleaved embryos were incubated in CR1aa medium containing 3 mg/ml FAF-BSA until day 4 at 38.8℃ in a 5% CO2 incubator. Embryos were then cultured in CR1aa medium containing 10% FBS until day 8. As a result, in vitro development rates were the highest in 350 ~ 400 kg body weight group and in 1++ grade group than other groups (p<0.05). However, there was no difference in in vitro developmental capacity of classified donor and non-donor oocyte groups. This result demonstrated that the better in vitro developmental capacity was obtained in high level originated oocyte groups (350 ~ 400kg, 1++ grade) than in others, while there was no different in donor types.

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.

Jeju Black Cattle (JBC) is an indigenous species of Korea and their mass production and industrialization are required for this high quality indigenous species. For production of elite JBC zygotes, selection of high quality sperm is necessary for in vitro fertilizatioin. In this study, we compared the sperm fertility and developmental capacity of IVF embryos using various JBC sperm (Bull A, B and C). The frozen semen was thawed and confirmed sperm viability and motility. In addition, frozen-thawed sperm was used for a chlorotetracycline(CTC) staining assay and in vitro fertilization. Sperm were classified into three staining patterns. The F pattern is indicative of uncapacitated sperm, the B pattern is indicative of capacitating and capacitated sperm and the AR pattern is indicative of acrosome-reacting sperm or acrosome-reacted sperm, respectively. Several kinds of JBC sperm was inseminated in 44 ㎕ IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 ㎕ heparin and 2 ㎕ PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. The sperm viability and motility were higher in sperm 3 species (n=8). When we confirmed sperm capacitation, F pattern and B pattern rate were higher than AR pattern in sperm A group. After IVF, the rates of cleavage and blastocyst development were higher in sperm C group compared to other sperm group. However, the cell number of blastocyst was higher in sperm E group. These results demonstrate that the use of sperm C was effective in production of elite JBC IVF embryos. Additional experimental data are required for more accurate analysis.

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 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.

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.

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