The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase (69.49 ± 3.16%) group than in the control (60.5 ± 3.94%) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine (17.12 ± 1.08%) group than in the control (21.46 ± 1.14%), catalase (20.54 ± 0.76%), and L-cysteine + catalase (19.29 ± 0.65%) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine (58.65 ± 1.39%) group than in the control (50.63 ± 2.37%) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine (3.74 ± 1.66%), catalase (4.65 ± 1.87%), and L-cysteine + catalase (8.11 ± 2.15%) groups than in the control (13.22 ± 1.6%) group (p<0.05). The glutathione level was significantly higher in the L-cysteine (1.33 ± 0.03%) group than in the control (1.08 ± 0.06 %), catalase (1.05 ± 0.02%) and L-cysteine + catalase (1.11 ± 0.03%) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein 50 μM for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and 50 μM and genestein 1 and 50 μM for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and 50 μM, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin 1 μM for 3 hr incubation. In mitochondrial activities, addition of quercetin 50 μM for 6 hr incubation increased mitochondrial activity but decreased at 100 μM concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin (1∼10 μM), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin (25∼50 μM). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin 10 μM (p<0.05). The developmental rates to morula or blastocysts at low (0.01∼1 μM) and high (5∼10 μM) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and 10 μM) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations (0.01∼10 μM).

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ( vs. , ) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

This study was carried out to effects of ethylene glycol concentration, sucrose and culture day of in vitro production embryo on slow-down freezing in Hanwoo. 6, 7, 8 and 9 day embryos produced in vitro were frozen using 1.8M EG+0.1M sucrose, 1.8M EG+0.5% BSA and 1.5M EG+0.1M sucrose media. Survivability was confirmed after frozen-thawed 24 and 48h and ICM, TE cell number were counted by Hoechst 33342 and PI staining after frozen-thawed 24h. As a result, 1.8M EG+0.1M sucrose group was most significantly (p<0.05) higher compared with the other treatment groups on survivability, TE and total cell number after frozen-thawed 24h (, and ). ICM number did not found significant (p<0.05) differences between the three treatment groups. in 6, 7, 8 and 9 day of embryos using three types of freezing media, frozen-thawed, 1.8M EG+0.1M sucrose groups with embryos cultured 8 day was significantly (p<0.05) highest survivability to after frozen-thawed 24h. 1.5M EG+0.1 sucrose group with embryos cultured 9 day was significantly higher survivability than group of embryos cultured 8 day after frozen-thawed 24 and 48h. In conclusion, 1.8M EG+0.1M sucrose media is considered to be effective to cryopreservation of embryos cultured 8 and 9 day.

The purpose of the present study was to investigate the effect of IFN- on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and concentration of progesterone (P4) in endometrial cells. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses of IFN- (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin and levels in the culture media were analyzed by enzyme immunoassays and total RNA was extracted from the cells for RT-PCR. P4 concentrations of blood samples were assayed by chemiluminescent immuno assays system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN- (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN- group (p<0.05). Although IFN- did not affect and production in epithelial cells, it decreased and production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN- (1 ug/ml). The results indicate that PG production was mediated by COX-2 expression in stromal cells but it was not affected in epithelial cells and this suggest that treatment of IFN- could improve the implantation environment of uterine by maintenance of high P4 concentration.

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.

BPA, a diphenyl compound containing groups, that make it structurally similar to synthetic estrogen and is considered as one of the major endocrine disruptors. Silymarin has extensively been used to prevent and/or alleviate some human disease, especially for the treatment of adverse liver conditions. It has an antioxidative efficacy and cancer preventive efficacy. Therefore, we examined the hypothesis that silymarin can inhibit BPA-induced toxicity in boar sperm duing in vitro storage. Sperm characteristics (motility, viability, membrane integrity and mitochondrion activity) in semen exposed to BPA (10~200 uM) were sharply lowered, while it increase in a dose and time dependent manner due to silymarin addition (50~200 uM) into semen extender in the presence of BPA (100 uM). All of the evaluated characteristics were gradually improved in the groups that were treated with silymarin (50~200 uM) in the presence of BPA (100 uM) in comparison to BPA 100 uM alone group, irrespective of incubation periods (3 and 6 h). These results demonstrate that silymarin can ameliorate the toxicity of BPA on boar sperm characteristics during in vitro storage, suggesting that silymarin indirectly act as an antioxidant.

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 MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to , and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.