After spermatogenesis, spermatozoa come in contact with fluids in the epididymis where they mature. During ejaculation, spermatozoa are mixed with secretions from prostate gland, vesicular glands, and bulbourethral glands. During natural mating, seminal plasma is deposited in the female reproductive tract eliciting various physiological and immunological responses. With the advances in proteomics, the components of seminal plasma have been identified and the information may be valuable in identifying markers for fertility. Components of seminal plasma that affect fertility have been discovered and the mechanism of action of these factors has been determined. The objective of this study was to determine the specific seminal plasma proteins from Korean native cattle, Hanwoo, and Korean native brindle cattle (KNBC) with the long term goal of improving fertilization rate. After SDS-PAGE and 2-dimensional gel electrophoresis, proteins were identified by Q -ToF analysis. They include plasma serine protease inhibitor precursor and platelet-activating factor acetylhydrolase after SDS-PAGE. Number and density of the spots in 2-dimensional gels were higher in KNBC than Hanwoo. Proteins identified from the paired spots of both breeds include chain A, bull seminal plasma PDC-109 Fibronectin Type II module, BSP-30 kDa precursor, and Spermadhesin Z13 or its precursor. Interestingly, some proteins were identified from multiple spots. The functional differences of these diverse forms of the proteins may require further studies. With their previously reported roles in sperm capacitation by these proteins, the studies on the mechanism of action, ligand interaction and the variation in the genome may help improving fertility in cattle.

This study evaluated a method of sorting X and Y chromosomes based on size using the forward angle light scatter related refractive index (FSC) of a flow cytometer. Hanwoo bulls sperm were separated to X and Y chromosomes by the parameters of FSC or Hoechst 33342 intensity. As a result, using monitor program linked flow cytometry during sorting processing, the purities were or for the X-fraction and or for the Y-fraction in the two sperm sorting methods. There were no differences in the X and Y ratios (X and Y %) between the sperm sorting methods based on FSC or DNA content. The proportions of female and male embryos used for in vitro fertilization and development were or , and or when sperm were processed using the sex sorting method by FSC or Hoechst 33342. In conclusion, further study is needed to determine the optimum procedure and improve the nozzle to enhancing sorting accuracy or efficiency. Also, the findings of this study do not negate the possibility that the difference method of sperm sorting cannot use a UV laser beam.

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability (84±1.15 %, p<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability (79±3 %, p<0.05) than those of the X and Y sperm (44.7±1.67 and 41.7±1.2 %) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities (24.3±1.2 and 25.7±0.9 %, p<0.05) compared to that of the sorted Y sperm (13.7±1.2 %) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher (55.0±1.7 and 45.0±1.5 %) than those of the sorted Y-sperm (32.3±0.9 %, p<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

Economic traits are quantitative traits and are mostly controlled by a large number of genes. Some these genes tend to have a large effect on quantitative traits in cattle and are known as major genes primarily located at quantitative traits loci (QTL). However, in practice, QTL is linked to allele associates of the gene controlling traits of interest. It is hypothesized that if QTL explaining a part of genetic differences between animals are detected, the effect of the genes located at QTL could assist in estimating an animal’s true genetic value. Therefore, QTL information could probably provides accuracy of breeding value estimation as well as more genetic gain through selection of animals at relatively younger age. Marker assisted selection (MAS) is the indirect selection process where a quantitative trait of economic importance is selected not just based on the trait itself but also on the basis of marker linked to QTL. MAS could be useful for traits that are difficult to measure, exhibit low heritability, and are expressed late in development. Major genes which are responsible for QTL could possibly be identified first by using different techniques such as gene expression analysis and QTL mapping. Thereafter, the information generated could be implemented for MAS in estimating breeding value. In this review we focused on delivering genome information into Hanwoo breeding program.

The Oct-4 (octamer-4), a member of the POU family transcription factor, is expressed in early mouse embryogenesis and in pluripotent embryonic stem (ES) lines. Oct-4 expression is thought to remain confined to the germline after gastrulation in the embryo. Therefore, the study was designed to, study the location of Oct-4 protein in the ovaries, placenta and testis of Korean native cattle (Hanwoo). Expression of Oct-4 mRNA in the ovaries and placenta of bovine was confirmed by RT-PCR and immunohistochemical analysis. Oct-4 was expressed in granulosa, thecal cells irrespective of the shape and size of follicles and endometerium of Korean native cattle (Hanwoo). Expression of Oct-4 was profound in all the tissues of Korean native cattle (Hanwoo) suggestung their role in them. Oct-4 localization and expression could contribute to further developmental studies in Korean native cattle (Hanwoo).

This study was performed to investigate the FSH levels for superovulation procedure in Korean Native Cattle (Hanwoo). The effectiveness of 200 mg and 400 mg of FSH to initiate superovulation was examined in Hanwoo. Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH group was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. Also, 400 mg FSH group was treated with 80, 60, 40, 20 mg FSH levels. On the 3rd day administration of FSH, 25 mg PGF2α was administered and CIDR was withdrawn. Donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 μg GnRH at time of 1st insemination and embryos were recovered 8 days after the 1st insemination. As a results, average number of CL treated with FSH 200 mg was higher as 20.9±1.20 than 15.8±0.63 for donors treated with FSH 400 mg, respectively(p<0.05). Treated group of 200 mg FSH level increased (p<0.05) the number of embryos recovered per procedure compared to 400 mg FSH level (18.2±1.18 vs. 12.38±0.52, respectively). When treatment of 200 mg FSH was performed, average transferable embryos/ova increased (p<0.05) to 14.1±1.12 from 6.8±0.33 of treated of 400 mg FSH. Group of 200 mg FSH increased (p<0.05) to 8.3±0.76 from 2.0±0.26 in morula stage compare to 400 mg FSH group. Mean of total early blastocyst and expanded blastocyst stage embryos was similar (p<0.05) between the 200 mg and 400 mg FSH levels group (4.7±1.19 vs. 2.9±0.18 and 1.2±0.40 vs. 1.9±0.17). These results suggest that 200 mg FSH level-based superovulation protocol with CIDR may be effectively used for production of superior embryos in Hanwoo. In other words, the less level of FSH may be effectively applied for Hanwoo (Korean Native Cattle), because Hanwoo was smaller body size than beef or daily cow.

포유동물 수정란의 동결보존기술은 최근 기후 변화에 따른 생물종 다양성을 보존하기 위해서 중요하게 여겨지는 연구 분야이다. 따라서 멸실 위험에 처한 동물의 개량과 증식, 보존과 복원 및 생명공학의 분야에 이르기까지 응용 기술은 다양하게 이용되어진다. 본 연구에서는 한우 수정란의 동결 후 생존성 향상을 위해서 동결 방법에 따른 체내 외수정한의 내동성을 조사하였다. 완만동결에 따른 체내 외수정란의 동결 융해 후 수정란의 재확장률은 89.6%와 81.5% 그리

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ( spermatozoa) at 4, 20 and 37. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38 was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37 in Hanwoo.

This study was performed in order to determine optimum flushing solution using the direct embryo collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3 day administration of FSH, 25 mg was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 GnRH at time of 1 insemination and embryos were recovered 8 days after the 1 insemination. Embryo collection from superovulated donors were performed to flushing by DEC and conventional method. As a results, the average number of recovered embryos were significantly higher as 19.11.40 with DEC method than 12.00.44 with conventional embryo collection method, respectively (p<0.05). Also, The average number of transferable embryos were significantly higher (p<0.05) as 15.81.72 with DEC method than 6.90.35 from conventional embryo recovery procedures. Meanwhile, number of recovered embryos and number of recovered transferable embryos following the number of flushing times until 6 flushing were significantly higher as 8.60.53 and 8.60.53 from 2 flushing time than other groups (p<0.05). No. of Ear. B stage embryos were significantly higher as 3.90.90 and 3.90.90 with 2 flushing time in total collected embryos and transferable embryos (p<0.05). Com M stage embryos were significantly higher as 3.71.00 in 2 flushing time and as 2.20.76 in 3 flushing time for recovered embryos (p<0.05). In transferable embryos, Com. M stage embryos were significantly higher (p<0.05) as 3.71.00 in 2 flushing time and as 2.20.76 in 34 flushing time, also. No. of degradation embryos was significantly higher as 2.20.72 in 5 flushing time, On the other hand, degradation embryos was not observed in transferable embryos (p<0.05). In conclusion, these results suggest that DEC method should effective methods for production of in vivo embryos using less flushing solution following perform until 4 flushing time than conventional embryo collecting method. Also, it might be effectively collection of transferable embryos following more less procedure times compared to conventional embryo recovery methods.

This study was performed in order to simplify the operation and minimize stress of donor and be readily available in the field with low cost and high quality embryos using the Direct Embryo Collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3rd day administration of FSH, 25 mg was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 GnRH at time of 1 st insemination and embryos were recovered 8 days after the 1st insemination. Embryo collection from superovulated donors was performed to flushing by non-surgical methods of 3-way, 2-way and DEC (l-way). The average number of recovered embryos were 11.250.63, 12.50.65 and 11.750.48 from operations of 3-way, 2-way and DEC methods, respectively. There were no significant differences among the embryo collection methods. Also, The average number of transferable embryos were 6.250.48, 7.250.48 and 7.250.63 from each embryo collection procedures. The number of transferable embryos was no differences among the 3-way, 2-way and DEC methods, respectively. Meanwhile, the ratio of transferable embryos for all recovered embryos from DEC methods was higher as 61.7 % than 55.6 %, 58 % from methods of 3-way, 2-way. And the flushing solution required for recovering embryos by DEC method was significantly lower as 0.280.32 1 than 1.80.12 1, 1.750.10 1 from 3-way, 2-way methods (p<0.05). Also, the time required for recovering embryos by DEC methods was significantly lower as 272 min than 513, 452 min, respectively (p<0.05). In conclusion, these results suggest that DEC method for embryo collection may be effectively used for production of in vivo embryos using less flushing solution and, it might be effectively available in the field compared to conventional embryo recovery methods using 3-way or 2-way balloon catheter.

This study was to evaluate the protein profile of seminal plasma using 2-DE in Hanwoo. Seminal plasma was harvested from five mature Hanwoo, and seminal plasma protein was extracted by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was . Immobilized pH gradient (IPG) strip was used 18 cm and 3~11 NL. SDS-PAGE was used 12% acrylamide gel. Each gels were visualized by comassie brilliant blue and silver staining. These spots were analyzed by MALDI-TOF MS and searched on NCBInr. The result, 20 proteins of 36 protein spots were searched through peptide sequencing on the NCBInr. 8 proteins profiled by 2-DE were proved through previous bovine studies and the name of each protein was albumin, nucleobindin, clusterin, TIMP-2, spermadhesin Z13, spermadhesin-1 and BSP proteins (BSP 30 kDa and BSP A1/A2). 12 new proteins were ATP synthase, protein MAK16 homolog, Transmembrane protein 214, E3 ubiquitin-protein ligase BRE1A, dual serine/threonine and tyrosine protein kinase, tissue factor pathway inhibitor 2, alpha-actinin-4, RUN domain-containing protein 3B, catenin alpha-1, protein-glutamine gamma-glutamyltransferase 2, plakophilin-1 and inter-alpha-trypsin inhibitor heavy chain H1 has not been previously described in the bovine seminal plasma study. These proteins may be contribute to define the type of proteins affecting fertility of male and improve the fertilizing ability of semen in Hanwoo.

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 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.