• Journal of Embryo Transfer
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• v.24 no.3
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• pp.225-230
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• 2009

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.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.777-784
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• 2006

This study was designed to investigate between the semen characteristics and sperm chromatin structure in boar with different farrowing rates and relationship between fertility by AI and results of sperm chromatin structure assay (SCSA). The CASA (computer-aided sperm analysis) and SCSA were performed with liquid semen in boars. The all SCSA parameters based on the farrowing rates by AI were significantly differ (P<0.05). The significant negative correlations (P<0.05) were observed between all SCSA parameters and farrowing rate obtained by AI in the field. In conclusion, these results suggest that the sperm parameters evaluated in these studies may be useful indicators to predict the fertility by AI.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.143-147
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• 2015

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen Post-thawed in boar. Recently, polymorphism (g. 35756 T>C) of Estrogen Receptor 1 (ESR1) gene reported to be significant association with MOT. This study was conducted to evaluate the ESR1 gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.35756 T>C SNP of ESR1 was significantly associated with frozen semen motility and kinematic characteristics. The g.35756 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.001). The SNP was also significantly associated with ALH (P<0.05). Therefore, we suggest that the g. 35756 T>C polymorphism in the intron 1 region of the porcine ESR1 gene could potentially be applied in frozen semen programs to improve MOT trait, but only after validation in other populations.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.163-166
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• 2012

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.97-104
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• 2008

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin. PA/plasmin system playa role in mammalian fertilization and motility and acrosome reaction of sperm. The present study was undertaken to identify PAs in porcine gametes and investigate a possible role of plasminogen in in vitro fertilization in the pig. When boar spermatozoa were preincubated in a fertilization medium (mTBM) for 0, 2, 4 or 6 h, the activity of tPA-PAI ($110{\sim}117\;kDa$), tPA ($62{\sim}70\;kDa$), and uPA ($34{\sim}38\;kDa$) was observed in the sperm incubation medium and sperm sample. PA activities in the sperm incubation medium significantly (p<0.05) increased according to increasing incubation times, while PA activities in sperm significantly (p<0.05) decreased at the same times. In addition, the rate of acrosome reaction in spermatozoa increased by increasing culture times. When oocytes were separated from porcine cumulus-oocytes complexes at 0, 22 or 44 h of maturation culture, no PA activities were observed in cumulus free-oocyte just after aspiration from follicles. However, the activity of tPA-PAI ($108{\sim}113\;kDa$) and tPA ($75{\sim}83\;kDa$) was observed at 22 h of in vitro culture and significantly (p<0.05) increased as the duration of the culture increased. On the other hand, when porcine oocytes were activated by sperm penetration or calcium ionophore, plasminogen significantly (p<0.05) increased ZP dissolution time (sec) in activated oocytes by sperm penetration. These results suggest that supplementation of plasminogen to fertilization medium may playa positive role in the improvement of in vitro fertilization ability in the pig.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.63-71
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• 2013

In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activity in post-thawed sperm that was supplemented with ginsenoside-$Rg_1$. Varying concentrations of ginsenoside-$Rg_1$ (0, 25, 50 and $100{\mu}M/ml$) were used in the extender during cryopreservation to protect the DNA of thawed sperm, thereby increasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. The results derived from CASA were used to compare the fresh, control, and ginsenoside-$Rg_1$ groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the $50{\mu}M/ml$ ginsenoside-Rg1 group ($61.0{\pm}4.65%$) than in the control ($46.6{\pm}7.02%$), $25{\mu}M/ml$ ($46.2{\pm}4.76%$), and $100{\mu}M/ml$ ginsenoside-$Rg_1$ ($52.0{\pm}1.90%$) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membrane integrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher ($91.6{\pm}0.82%$) in the $50{\mu}M/ml$ ginsenoside-$Rg_1$ group than in the other groups. Here, we report that ginsenoside-$Rg_1$ affects the motility and viability of boar spermatozoa. Moreover, ginsenoside-$Rg_1$ can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

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 ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, 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.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.263-266
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• 2010

Prediction of semen's fertilizing ability used in artificial insemination (AI) is one of very important factors on pig reproductive performance. In vitro fertilization (IVF) has been used for indirect evaluation of sperm's fertilizing ability and it has been showed as highly correlated index. In swine industry, increasing interest in preservation of boar semen raises questions on the sperm motility from semen used in commercial AI centers. Mitochondria in sperm mid-piece generate the energy to support motility and could be an explanation of impaired fertility. Objective of this study was to suggest usable sperm motility to farms in measuring the effect of sperm motility and sperm abnormality on in vitro production of embryo in which sperm's fertilizing ability can be determined indirectly. Semen samples were provided from local AI center and used within 3 days after collection. Semen samples were divided by 4 different motile groups (>70%; 61~70%; 51~60%; <50%) using CASA (computer-assisted sperm analysis) on the days of IVF. Developmental rate to the blastocyst stage from over 61% motile sperm group showed significantly higher rate than below 60% motile sperm group ($16.5{\pm}0.7{\sim}18.4{\pm}0.8%$ vs $6.3{\pm}0.8{\sim}11.5{\pm}0.7%$, p<0.05). In experiment to determine the relationship between sperm motility and viability and abnormality, over 61% motile sperm groups showed significantly higher viability rate compared to below 60% motile sperm groups ($84.8{\pm}4.0{\sim}88.1{\pm}4.0%$ vs $69.1{\pm}4.0{\sim}74.2{\pm}4.0%$, p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ($10.2{\pm}2.2$ vs $16.0{\pm}2.2{\sim}21.0{\pm}2.2%$, p<0.05). In experiment to find the correlation between sperm motility of 4 different motile groups and amount of mitochondria, lower motility group also showed lower level of mitochondria (p<0.05). The mitochondria parameter used in this study showed another possibility to differentiate the sperm motility. Taken together, because below 60% motile semen used in AI reduce the fertility, AI centers should provide the over 60% motile sperm to the farms at the time of AI.

• Korean Journal of Agricultural Science
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• v.47 no.3
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.263-273
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• 2002

This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.