This study investigated the effects of L-carnitine (LC) and nicotinic acid (NA) on sperm viability during liquid storage at 18℃ in miniature pigs. 10 μM LC and 30 mM NA, combined LC and NA (LN) were treated in fresh semen for 3, 7, and 10 days. In results, sperm survival increased in NA- and LN-treated semen on 7 and 10 days (p<0.05), mitochondrial integrity of live sperm increased in LN-treated semen on 7 days (p<0.05), but not NA-treated semen. In addition, we examined the acrosome reaction of sperm in miniature pigs. LC and NA did not influence on acrosome reaction of boar sperm. In conclusion, LC and NA effectively maintained the viability and quality of sperm during long-term storage in miniature pigs, suggesting that the combined LN may be useful for improving the semen extender for long-term liquid storage in pigs.

The objective of this study was to investigate the efficiency of nicotinic acid during in vitro fertilization (IVF) in frozen-thawed bull sperm . The ejaculated semen was diluted with Triladyl containing 20% egg-yolk and cryopreserved in liquid notrigen. The frozen sperm was thawed for 45 seconds in the 38℃ water bath. Sperm was diluted with IVF medium (Bovine-Oviduct medium; BO) containing 0, 15, 30 and 60 mM nicotinic acid (NA), which were incubated at 39℃, 5% CO2 for 0, 0.5, 1, 2 and 4h. The characteristics of frozen-thawed sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI for outer acrosomal membrane damage and Rhodamine123/PI for mitochondrial integrity using flow cytometry. And the sperm ability was analysed by Coomassie brilliant blue (CBB) staining for acrosome reaction state and Rose bengal staining for abnormality. Acrosome reaction and abnormality were analyzed using a microscope. In results, sperm viability was significantly higer in 30 mM group than 0 and 15 mM groups at 1 and 2 h (p<0.05). Outer acrosomal membrane damage was significantly lower in 30 mM group than 0 and 15 mM groups at 1, 2 and 4 h (p<0.05). And mitochondrial integrity was significantly higher in 30 mM group than 0 and 15 mM groups at 2 and 4 h (p<0.05). Also, acrosome reaction was significantly lower in 30 mM than 0 and 15 mM groups at 1 and 2 h (p<0.05) and abnormality was lower NA groups than 0 group at 1 h (p<0.05). In couclusion, we suggest that using the thawing medium containing NA for sperm dilution can be benefical for IVF in bulls

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM (61.1 ± 1.5%, 64.7 ± 2.0%) of nicotinic acid than other groups (0 mM, 52.1 ± 2.3%; 20 mM, 47.8 ± 5.1%, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid (26.1 ± 1.8%, 24.9 ± 1.5%) than other groups (0 mM, 35.3 ± 0.8%; 20 mM, 36.5 ± 1.9%, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM (84.2 ± 3.6%, 88.4 ± 2.3%) of nicotinic acid than other groups (0 mM, 77.3 ± 4.4%; 20 mM, 73.3 ± 3.6%, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM (17.0 ± 1.3%) of nicotinic acid than other groups (0 mM, 9.4 ± 0.5%; 5mM, 12.6 ± 0.8%; 20 mM, 5.0 ± 1.0%, P<0.05). Moreover, total cell number was higher in 5 and 10 mM (53.6 ± 2.9%, 57.9 ± 2.8%) of nicotinic acid than other groups (0 mM, 41.0 ± 1.4%; 20 mM, 23.2 ± 2.8%, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid (0.7 ± 0.1%) than other groups (0 mM, 1.0 ± 0.1%; 10mM, 0.9 ± 0.0%; 20 mM, 1.4 ± 1.0%, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

Objective of this study was to investigate the effect of nicotinic acid (NA) on the characteristics in fresh semen of miniature pig. We evaluated viability, acrosome reaction and mitochondrial integrity of sperm on 0, 3, 7 and 10 days during storage period with nicotinic acid. As results, the survival rate of sperm in 15 mM NA (day 3, 87.8 ± 1.2%; day 5, 84.0 ± 2.7%; day 7, 82.2 ± 0.9%) and 30 mM NA (day 3, 87.7 ± 0.3%; day 5, 84.4 ± 2.5%; day 7, 82.3 ± 0.7%) groups were higher than control and 5 mM NA groups in 3, 7 and 10 days of semen storage. The NA-treated sperm on 10 day was used day for observing acrosome integrity. The survival sperm with acrosome reaction was higher in 30 mM NA group (day 3, 2.7 ± 0.2%; day 5, 3.3 ± 0.6%; day 7, 11.4 ± 0.3%) than in the control, significantly (P<0.05). Moreover, the live sperm with mitochondrial integrity was higher in whole treatment groups of NA than control group, significantly (P<0.05). Specially, most mitochondrial integrity on 10 day of semen storage was significantly higher in 30 mM NA group (90.2 ± 1.6%) than other treatment groups (control, 81.8 ± 3.1%; 5 mM NA, 83.4 ± 3.0%; 15 mM NA, 89.1 ± 0.7%, P<0.05). In conclusion, supplement of NA in liquid semen of miniature pig can improve and maintain semen quality, such as viability, acrosome reaction, and mitochondria integrity.

L-Carnitine is an antioxidant for the transport of fatty acids in mitochondria and breakdown of lipids for metabolic energy. Some studies have suggested that carnitine improves sperm motility in mammals. The objective of this study was to investigate the effect of L-carnitine on the characteristics in fresh semen of miniature pigs. The collected fresh semen was stored in modena B medium with L-carnitine (0, 1.0, 2.0, and 4.0 mg/ml) for 10 days at 18℃. The semen quality of viability, acrosome reaction and mitochondria integrity was analyzed on 0, 3, 7, and 10 day of semen storage. The percentages of live and dying sperm were not different among treatment groups with different concentrations of L-carnitine during the storage period. In acrosome reaction analysis, when the sperm stored for 7 day, the percentages of live sperm with acrosome reaction were significantly (p<0.05) lower in 1 (9.0±0.9%), 2 (7.6±0.2%) or 4mg/ml (7.9±0.8%) L-carnitine-treated groups than the control group (0 mg/ml L-carnitine) (11.12±0.2%). However, there were no difference in percentages of live sperm with acrosome reaction for 3 and 10 days of storage with each concentrations of L-carnitine. When sperm was stored for 3 and 10 days, the percentages of live sperm with mitochondria integrity were significantly higher in 2 mg/ml of L-carnitine-treated group than control group (p<0.05). In conclusion, the L-carnitine has a positive effect on acrosome reaction and mitochondria integrity in liquid state of fresh semen in miniature pigs.

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTrackerTM Red, DC-FDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ≥16 cell embryos were significantly different in fore type oil treat-ment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treat-ment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.

The objective of this study was to investigate the relationship between fertility and protein pattern change using in vitro fertilization, analysis of sperm characteristics and two-dimensional gel electrophoresis in different pig types. In results, the viability and mitochondria integrity of sperm were higher significantly (p<0.05) but the portions of ac-rosome reaction was lower significantly (p<0.05) in Duroc and F1 (potbellied×PWG miniature pig) than PWG minia-ture. On in vitro fertilization to investigate fertility, the fertility of F1 semen war higher significantly (p<0.05) than in Duroc and PWG miniature pig. On the other hand, protein patterns showed similar function among the different boar semen. Especially, the heat shock 70 kDa 1-like and G patch domain-containing protein 4 were significantly (p<0.05) higher expressed in F1 than in Duroc and PWG miniature pig. The proteins associated with mitochondria in Duroc were significantly (p<0.05) higher expressed than in F1 and PWG miniature pig. The developmental rates to blastocyst stage of oocytes fertilized with sperm of F1 pig were significantly (p<0.05) higher than in PWG miniature pig. However, phosphoglycerate kinase 2 and zinc finger protein 431 were significantly (p<0.05) higher expressed in PWG miniature pig than in F1 and Duroc pigs. In conclusion, the results of the present study indicate that different proteins were expressed in different pig types, and were associated with a sperm functions and embryo develop-ment.

An uterus is female reproductive tract organ that affected estrus cycle. During a various changes occur at uterus in estrus cycle, one of them is body fluids secretion be called uterine fluid. Therefore, the objective of this study was to investigate the changes of protein patterns using two-dimensional gel electrophoresis in uterus fluids during the follicular and luteal phases in estrus cycle of pigs. In changes of protein spots were confirmed during the follicular and luteal phases. The 136 spots were expressed in follicular phase, the 57 spots of them showed reproducibility. On the other hand, the 140 spots were expressed in luteal phase, the 73 spots of them showed reproducibility. Also, spots expressed in follicular phase were number 69 and 94 spots and spots expressed in luteal phase only were number 156, 157, 184～187, 190 and 191 spots. The spots which of higher expression levels in the luteal phase than in follicular phase were number 76 and 79 spots. In conclusion, the spots expressed in follicular and luteal phases were confirmed with difference levels and these differences are function of RNA resolving, protein synthesis and cytoskeletal architecture.

Buckwheat (Fagopyrum esculentum Moench), one of the minor crops grown in Korea belonging to the Polygonaceae family, is an annual crop widely cultivated in Asia, Europe, and America and has a character of outcrossing and self-incompatibility. The objective of this study was to analyze the genetic variability, phylogenetic relationships and population structure of buckwheat landraces of Korea using SSR markers. Ten microsatellite markers have been detected from a total of 79 alleles among the 179 buckwheat accessions were collected from Korea. The number of allele per marker locus (NA) ranged from 2 (GB-FE-001, GB-FE-043 and GB-FE-055) to 31 (GB-FE-035) with an average of 7.9 alleles. GB-FE-035 was the most polymorphic with the highest PIC value 0.93. Major allele frequencies (MAF) for the 10 polymorphic loci varied from 0.12 to 0.97 with a mean allele frequency of 0.57. The expected heterozygosity (HE) values ranged from 0.05 to 0.94 with an average of 0.53. The observed heterozygosity (HO) ranged from 0.06 to 0.92 with an average of 0.42. The overall polymorphic information contents (PIC) values ranged from 0.05 to 0.93 with an average of 0.48. The landrace accessions of buckwheat used in the present study were not distinctly grouped according to geographic distribution. The study concludes that the results revealed genetic differentiation was low according to the geographic region because of outcrossing and self-incompatibility. We reported that our analyses on the genetic diversity of common buckwheat cultivars of Korea were performed by using of microsatellite markers.