Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.

The aim of the present study was to identify coat color associated genes that are differentially expressed in mature Korean brindle cattle (KBC) with different coat colors and in Hanwoo cows. KBC calves, before and after coat color appearance, were included. Total cellular RNA was isolated from the tail hair cells and used for microarray. The number of expressed coat color associated genes/probes was 5813 in mature KBC and Hanwoo cows. Among the expressed coat color associated genes/probes, 167 genes were the coat color associated genes listed in the Gene card database and 125 genes were the pigment and melanocyte genes listed in the Gene ontology_bovine database. There were 23 genes/probes commonly listed in both databases and their expressions were further studied. Out of the 23 genes/probes, MLPH, PMEL, TYR and TYRP1 genes were expressed at least two fold higher (p<0.01) levels in KBC with brindle color than either Hanwoo or KBC with brown color. TYRP1 expression was 22.96 or 19.89 fold higher (p<0.01) in KBC with brindle color than either Hanwoo or KBC with brown color, respectively, which was the biggest fold difference. The hierarchical clustering analysis indicated that MLPH, PMEL, TYR and TYRP1 were the highly expressed genes in mature cattle. There were only a few genes differentially expressed after coat color appearance in KBC calves. Studies on the regulation and mechanism of gene expression of highly expressed genes would be next steps to better understand coat color determination and to improve brindle coat color appearance in KBC.

Amphiregulin (AREG), a glycoprotein that is a member of the epidermal growth factor (EGF) family, is expressed by the porcine conceptus and endometrium. AREG genotypes were determined based on an SNP in the intron 3 of the gene. Contradictory effects of AREG genotypes on reproductive traits in different pig breeds were reported previously. G allele had undesirable effect on reproductive trait in Meishan breed, while it had favorable effects in Polish Landrace and Large White. We determined AREG genotypes of 179 pigs including the Duroc, Landrace, Yorkshire, Korean native pig (KNP), and Meishan breeds. Two new SNPs were identified near the previously reported SNP in the intron 3 of AREG. Frequencies of AREG alleles among the Duroc, Landrace, Yorkshire, and KNP sows were significantly different (p<0.001), indicating association between AREG genotypes and pig breeds. The first parity litter size was significantly affected by the breeds (p=0.014), but not by AREG genotypes (p=0.148). However, there were breed and AREG genotype associated trends in the first parity litter size. The first parity litter size appeared to be higher in Duroc and KNP sows with G allele, while it appeared to be lower in Landrace sows with G allele. Significant variability of AREG alleles among pig breeds, for the first time in Duroc and KNP sows, was identified. AREG genotypes may influence reproductive traits differentially for each breed and thus, AREG genotypes may need to be considered when sows are bred to increase litter size.

The aim of this study was to determine the relationship between the coat color appearance of Korean brindle cattle and the changes of relevant hormone levels that may affect the hair pigmentation during different stages of growth and maturation. In mature cattle, levels of both ACTH and DHEA in Korean brindle cattle with brown color were significantly higher than those with black color (p<0.05). Levels of α-MSH in Korean brindle cattle with whole brindle (≧50%) color were significantly higher than those with brown color (p<0.05). In calves of Korean brindle cattle at 2 to 6 months, the concentration of estradiol was significantly higher in calves with whole brindle color than those with part brindle color (p<0.05), when the coat color was confirmed. After 6 month of coat color confirmation, levels of testosterone and ACTH increased in calves with part brindle color and were significantly higher than those with whole brindle color (p<0.05). In calves of Korean brindle cattle at 1 or 2 months, there were no significant differences in hormone levels of estradiol, ACTH, DHEA and α-MSH between the calves with brindle color and brown color, except estradiol before brindle color appearance. Changes of relevant hormone levels at different stage of growth and maturation may affect the pigmentation of coat during the development of cattle. In addition to the current study correlating the different coat colors with relevant hormone levels, investigation of the coat color associated genes expressed in Korean brindle cattle may further clarify the mechanisms of coat color changes during their development.

The objective of this study was to determine the effect of the MC1R genotypes of the Chikso (Korean brindle cattle) sires on the coat colors of their offspring. In this study, 15 Chikso sires with known MC1R genotypes were used for breeding in the Gangwon Province Livestock Research Center, the Chungbuk Institute of Livestock and Veterinary Research, and the Livestock Experiment Station, Jeonbuk Institute of Livestock and Veterinary Research from either 2011 or 2012 to 2013. There were 6 sires with E+E+ genotypes and 9 sires with E+e genotypes, and their coat colors were all whole brindle (more than 50 of the body). Among the 90 calves produced in 2011∼2013 or 2012∼2013 from the 15 sires, 50 (55.6%) of them were females and 40 (44.4%) of them were males. Coat colors of the offspring were determined when they reached over 6 months of age. Calves with whole brindle, part brindle, brown and black coat colors were 42 (48.3%), 11 (12.6%), 18 (20.7%) and 16 (18.4%), respectively. Ratio of calves with whole brindle coat color was higher than any other coat colors. Among the offspring with whole brindle color, 20 (41.7%) calves were female and 22 (51.3%) calves were male. By determining the MC1R genotypes of the dams and calves in this study along the family lines, and investigating other genes that may be involved in the coat colors of the Chikso, better breeding system may be established to increase the brindle coat color appearance in the future.

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

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.

Plant biomass is a huge carbon-complex that has potential as a nutrient. Therefore we extracted and separated useful materials for plant growth from tea leaf and stem. The pre-treatment process including high temperature (200 °C) and pressure (20-40 kgf/cm2) was treated for several minutes and extracted at 120 °C for 30-60 minutes. After that the chemical compositions and ingredients were analyzed from that plantnutrient. As a result of mineral contents, calcium and magnesium concentrations are higher than other minerals. Also the result of carbohydrates analyses has shown that the sugar oligomer consists of xylose(95.3%) and glucose(4.7%), and the sugar monomer consists in the order of xylose (52.7%) > manose (22.8%) > arabinose (10.8%) > galactose (10.2%) > glucose (3.5%). Before applied to field, in vitro plant growth system and formulation were examined. To evaluate the effect of the nutrients, both strawberry green-house and persimmon fields were used in this test. The treated persimmons were heavier than controls scored at 13-22%. In addition, the storageperiod was extended in the treated strawberries. Interestingly in the treated strawberry, the contents of polyphenols were increased (38-57%). These results suggest that the plant-nutrient can afford to help for plant growth and storage, and it can be substituted for other commercial nutrients. In conclusion, this plant-nutrient may help to extend eco-friendly or organic farming in Hadong-gun area.