There are four (yellow, brindle, black, black in Jeju) kinds of native cattle in Korea. But only twelve hundred Korean brindle cattle (KBC) are remaining in limited areas of Korea and the genetic lineage, diversity, polymorphisms of KBC has not been identified. To analysis genetic polymorphism of KBC, 33 KBC were characterized using 11 microsatellite markers. Size of microsatellite marker was decided using Gene Mapper software after analysis ABI 3130XL. The average of allele numbers of KBC was 6.7 in this study, but that of Hanwoo was 10.0 in our previous report. The observed and expected heterozygosities of KBC were 0.719 and 0.738 but those of Hanwoo were 0.751 and 0.760 in our previous report. Also polymorphism information content (PIC) values were average 0.690 in KBC but 0.725 in Hanwoo. These results demonstrate that genetic polymorphism of KBC have decreased because the population was limited.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was performed to investigate whether oxygen consumption reflects morphological grade of in vivo derived bovine blastocyst-stage embryos (blastocyst). The oxygen consumption of in vitro produced blastocyst was compared to its total cell number. In addition, pregnant rate was measured after transplantation of in vivo blastocysts with different oxygen consumption. The quality of blastocyst collected on day 7 after artificial insemination was categorized as grade I and II (G I and G II) based on microscopic observation of the morphology. Oxygen consumption of blastocyst was measured using a scanning electrochemical microscopy (SECM) and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide. Pregnancy of recipient cow was confirmed with rectal palpation after 60 days of embryo transfer. The oxygen consumptions of G I blastocysts were significantly higher than those of G II blastocysts ( versus , p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7, and 110.2 in the oxygen consumption of below 10.0, 10.0~12.0, and over respectively. Total cell number was significantly increased in embryos with high oxygen consumption (p<0.05). Pregnant rate in recipient cow was 0, 50, and 85.7% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0, and over , respectively. These results suggest that measurement of oxygen consumption may help increase the pregnant rate of bovine embryos.

난포낭종은 소 번식 장애의 주요 원인 중의 하나이며, 다양한 유전자의 변화는 여러 세포와 조직 기능에 영향을 준다. 이러한 유전자 변화는 낭종성 난소에서도 나타날 수 있다. 이온 및 수송체와 관련된 유전자 변화가 한우의 난포낭종을 유발할 수 있을 것이라는 가설 하에 난포낭종성 난포에서 발현 변화를 보이는 유전자를 찾기 위하여 마이크로어레이 분석을 수행하였다. 마이크로어레이 분석 결과, 난포낭종성 난포에서 FGG와 LRP8이 증가하고, SLC44A4, S

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen‐thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2‐cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time‐dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption of embryos. Here, we measured oxygen consumption of bovine embryos at various developmental stages was measured using a scanning electrochemical microscopy (SECM). We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell-stage to morula-stage), indicating that oxygen consumption reflects the cell number ( versus , p<0.05). In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos ( versus , p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro-derived bovine blastocyst-stage embryos (p>0.05). In the frozen-thawed blastocyst-stage embryos, live embryos showed significantly higher oxygen consumption than dead embryos ( versus , p<0.05). These results indicate that the measuring oxygen consumption by SECM can be used to evaluate bovine embryo quality.

We separately cultured follicular oocytes collected from individual ovaries of slaughtered Korean native cows and examined both the embryonic development rate and pregnancy rate after embryo transplantation according to the meat yield and quality grades of the source beef carcass. Oocytes from meat yield grade B cows exhibited a higher fertilization rate and embryonic developmental rate to the eight-cell stage than oocytes from grade A or C animals (p<0.05), but there was no significant difference in rate of development to the blastocyst stage among meat yield grades A, Band C. The oocyte cleavage rate and development rate to the eight-cell stage from meat quality grade 3 cattle was higher than grades 1++, 1+, 1 and 2 (p<0.05). Embryos derived from grade animals displayed a development rate to the blastocyst stage of 19.4%, which was also higher than all other meat quality grades (p<0.05). Transplantation of in vitro-cultured oocytes from meat yield grade A ovaries led to a higher pregnancy rate (64.2%) than in vitro-cultured oocytes from meat yield grade B ovaries (56.5%), but there was no significant difference between the two groups in pregnancy or abortion rates. In conclusion, embryonic development rate and pregnancy rate has a close relation to meat quality grades of the source beef carcass, this results is to give information for the Korean native cows improvement of breed.

The aim of the present recent study was to compare the protein patterns in the vaginal mucus of Hanwoo cattles during spontaneous and CIDR induced-estrus. Ten cattles, who had been observed in estrus, received no treatment and served as the group of cattles with normal spontaneous estrus. Thirteen cattles in the CIDR received an CIDR insert on day 14 were removed and cattles were injected GnRH on day 15. Vaginal mucus samples were collected from all cattles at the same time the single AI in cattles with spontaneous estrus and the AI in cattles with induced estrus. Spontaneous and CIDR-induced estrus vaginal mucus samples were analyzed on two different array surfaces: cation-exchange (CM10), anion-exchange (Q10). In addition, using the NaCl solution by which the proteins combined after washing are 0.5, 1 and 2 M, it was fractionated and a protein was collected successively. The results are summarized as follows: 1) Ionic surfaces chemistries (Q10 and CM10) gave the best results in terms of detectable protein peaks, with more than 100 protein peaks in the two fractions and under each condition. 2) Protein mass spectrometer using 11 different proteins in protein identification of 7 were able to determine the protein. List of identified proteins as follows; Ribosome-binding protein 1, GRIP 1-associated protein 1, Katanin p60 ATPase-containing subunit A-like 1, Protein FAM44A, DUF729 domain-containing protein 1, Prolactin precursor, Dihydrofolate erductase. Conclusively, on the basis of this study, protein expression in the vaginal mucus could be used as an indicator for time of estrus manifestation in order to increase conception rates by applying AI at an optional time.

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes (156.0+-68.2 vs 134.7+-54.8)). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total (160.0+-69.0 vs 156.7+-68.4) and apoptotic cells (14.0+-10.4 vs 11.8+-6.4)) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

This study was carried out to investigate inflammation-related gene expression altered in ovary and endometrium of Korean cattle with reproductive disorders using microarray. In the present study, nine inflammation-related differential1y expressed genes (DEGs) were identified in the cystic ovary and endometrium with endometritis. In the follicular cyst, eotaxin and alpha-2-HS-glycoprotein (AHSG) were up-regulated, whereas complement component 3 (C3) and oxidised low density lipoprotein (lectin-like) receptor 1 (OLR1) were down-regulated. Complement component 4A (C4A) was up-regulated in luteal cyst. In the endometritis, chemokine 1igand l and 2 (CXCL1 and CXCL2), protein C (inactivator of coagulation factors Va and VIIIa), and complement component C5 were up-regulated, whereas kininogen was down-regulated. Of these genes, we focused on eotaxin and kininogen, which were highly regulated in the follicular cyst and endometritis, respectively and on C3 commonly regulated in both reproductive disorders. The microarray data of eotaxin, kininogen, and C3 were validated by semi-quantitative PCR. Consistent with microarray data, eotaxin was up-regulated by 4-fold in the follicular cyst, while kininogen was down-regulated by 5-fold in the endometritis. C3 was down-regulated in the both follicular cyst and endometritis. Our results suggest that these inflammation-related genes could be useful markers for diagnosis of cystic ovary and endometritis of Korean cattle.

The objective of this study is to investigate the changes in concentrations of leptin and insulin in serum of Korean cattle (Hanwoo) with reproductive disorders and to examine the relationship among leptin, insulin, and body condition score (BCS). The concentration of leptin in serum of pregnant Hanwoo showed insignificant difference from that in serum of Hanwoo with reproductive disorder, such as repeat breeding, follicular cyst, corpus luteum cyst, ovarian atrophy, and feeble estrus (p>0.05). However, the concentrations of leptin and insulin in serum were changed with different BCS value. In emaciated Hanwoo (BCS ), they were significantly decreased compared to BCS (p<0.05). The leptin showed different genotypes with different BCS value. In BCS , C/T genotype was expressed (83.3%) more than C/C (16.7%) or T/T (0%) genotype, whereas C/C genotype was expressed (62.5%) more than C/T (25.0%) or T/T (12.5%) genotype in BCS . The insulin concentration in follicular fluid obtained from ovary with follicular cyst which has follicles having diameter of was significantly higher (p<0.05) than those in normal follicle fluid which has follicles having diameter of . These results showed that concentration of leptin and insulin in serum were related to BCS value and follicular size and suggest that the changes in concentration of leptin and/or insulin in serum could be a potent biomarker for diagnosis of bovine reproductive disorder.

This study examined pregnancy and fetal loss rates according to different estrus synchronization protocols and injection of gonadotropin releasing hormone (GnRH) after transfer of Korean Native Cattle embryos to Holstein recipients. In Experiment 1, recipients received no treatment (Control, n = 119); two injections of prostaglandin ( ) 11 days apart (PGF group, n = 120); GnRH (day 0)- (day 7)-GnRH (day 9) (Ovsynch group, n = 120); and CIDR (day 0)- and CIDR removal (day 7)-GnRH (day 9) (CIDR group, n = 110). In Experiment 2, the control group was received no treatment of GnRH. The treatment groups were received GnRH at embryo transfer (ET) (day 0), 7 days later, 14 days later, ET and 7 days later, 7 and 14 days later, or ET, 7 and 14 days later. Recipients were assigned to treatment randomly and received two in vitro produced blastocysts. Pregnancy was diagnosed at day 60 by palpation per rectum. Fetal loss to term was determined by palpation every 90 days thereafter. In Experiment 1, the pregnancy rate in the CIDR group (59.1%) were higher than in the Control group (42.0%) (p<0.01); fetal loss rates were similar for all groups (12.0 to 18.5%). In Experiment 2, the pregnancy rate in Day 0+7+14 group was higher (60.2%) than the control (40.2%) (p<0.01) and resulted in a lower fetal loss (p<0.05) than the control (4.6 vs. 11.4%). There were no significant difference between other treatment and the control (p>0.05). These results show that pregnancy rates of bovine embryos can be enhanced by CIDR insertion or GnRH treatment. Additionally, fetal loss may be reduced with GnRH treatment after ET.

This study compared the pregnancy rates of Korean native donor cattle after either a timed artificial insemination (TAI) or embryo transfer (TET) following the synchronization of ovulation using a controlled internal drug release (CIDR) device together with estradiol benzoate (EB) and prostaglandin (). Fifty five cows and 8 heifers which had been previously used for embryo production were assigned to two treatments: (1) Thirty-two cattle received a CIDR device and 2 mg EB (Day 0), 25 mg injection at the time of CIDR removal on Day 7, and 1 mg EB injection on Day 8. All of the cattle received a TAI 30 h (Day 9) after the second EB injection (TAI group). (2) Thirty-one cattle received the same hormonal treatments as in the TAI group. The cattle with corpus luteum (CL) received a TET on Day 16 using frozen-thawed embryos (TET group). Ultrasonographic observations demonstrated that the proportion of cattle with synchronized ovulation on Day 10 and the concomitant formation of new CL on Day 13 did not differ between groups (p>0.05); the overall mean rates were 65.1 and 73.0%, respectively. The conception and pregnancy rates did not differ (p>0.05) between the TAI (12.5% and 12.5%) and TET groups (13.0% and 9.7%), respectively. We conclude that the pregnancy rate following TAI or TET in Korean native donor cattle was poor, which might be due in part to a poor synchrony of ovulation and concomitant CL formation.

The objective of this study is to investigate the changes in concentrations of blood urea nitrogen (BUN) and sex steroid hormones, such as estrogen, progesterone, and testosterone in Korean cattle (Hanwoo) with reproductive disorders and to examine the relationship between BUN and body condition score (BCS) in Hanwoo. The concentration of BUN was 16.2 mg/dl, 17.8 mg/dl, 15.1 mg/dl, 17.9 mg/dl, and 28.3mg/dl in pregnancy, repeat breeding, follicular cyst, luteal cyst, and ovarian atrophy, respectively. In Hanwoo with BCS , and , the concentration of BUN was 15.8 mg/dl, 17.0 mg/dl, and 17.6 mg/dl, respectively. Fluoroimmunoassay showed that serum estrogen and progesterone levels were decreased in reproductive disorders Hanwoo, such as ovarian atrophy, endometritis, and weak estrus. The testosterone level was significantly decreased in Hanwoo with reproductive disorders compared to that in pregnant Hanwoo ( vs 0.13 ng/ml, p<0.05). The progesterone and estrogen concentrations in follicular fluid obtained from ovary with follicular cyst were significantly higher (p<0.05) than those in normal follicle fluid. These results show that there is no relationship between BUN and BCS in Hanwoo, and the concentration of sex steroid hormone in serum and follicular fluid are changed in reproductive disorders Hanwoo.

This study was performed to identify and analyze the specifically expressed plasma proteins during early pregnancy in both pregnant and non-pregnant Hanwoo. Blood samples were collected at 0 (the day of AI), 2, 3, 4, 7, and 11 weeks after AI from pregnant (n=3) and non-pregnant (n=4) Hanwoo, respectively. The hematological parameters were measured. After 2-dimensional electrophoresis using serum, normalized protein spots were selected for the significant expression variation deviated over two fold in its expression level between two groups. Among 17 spots selected, 15 were identified as albumin, IgG1 heavy chain constant region, haptoglobin, ferrochelatase, fibrinogen, hemopexin. 5 spots were expressed only in non-pregnant specific. The spot identification of 1105 and 6106 was decreased after 3 weeks from AI. However, 2/17 spots were still unidentified. Further studies are needed to analyze the function of the proteins associated with early pregnancy.