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. Estrogen receptors 2(ESR2) is involved in estrogen related apoptosis in cell cycle spermatogenesis, but their functions have not been confirmed in pig until now. Therefore, this study was conducted to analyze their association with sperm motility and kinematic characteristics. DNA samples from 105 Duroc pigs with records of semen motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were analyzed. A SNP in coding region of ESR2 g.35547A > G in exon 5 was associated with MOT (p < 0.05) in Duroc population. Therefore, we suggest that the porcine ESR2 gene may be used as a molecular marker for Duroc boar semen quality, although its functional effects were not defined yet. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate gene for boar fertility, but still the lack of association across populations should be considered.

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as a candidate gene to investigate Duroc boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT, 82.27±5.58), Curvilinear velocity(VCL, 68.37±14.58), Straight-line velocity(VSL, 29.06±6.58), the ratio between VSL and VCL(LIN, 47.36±8.42), Amplitude of Lateral Head displacement(ALH, 2.88±0.70)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p<0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

The objective of this study was to compare the effect of semen extenders on the sperm motility, viability, acrosome integrity and functional integrity of plasma membrane (HOST: hypo-osmotic swelling test) during liquid preservation of Korean Native boar semen. In this experiment, semen was diluted in Androhep plus, Beltsville Thawing Solution (BTS), ModenaTM, Seminark and Vitasem LD. Sperm-rich fractions were collected from three Korean Native boars and sub-samples were diluted (30×106 spermatozoa/ml) in different semen extenders. Semen samples were stored at 17℃ for 96 hours. On everyday (0, 24, 48, 72, 96 h) after storage, the sperm characteristics relevant for fertility, such as sperm motility, viability, acrosome integrity and HOST positive were evaluated. The motility of spermatozoa stored in different extenders was no significantly different among other extenders (P>0.05). Also, no difference was observed among samples processed with different extenders in the percentage of sperm viability, acrosome integrity and HOST positive. All extenders maintained a high percentage (70%) of sperm motility, viability and acrosome integrity through 96 h of storage. The result of this study show that there was no significant differences among extenders in their capacity to preserve motility, viability and membrane integrity of spermatozoa from normal, fertile Korean Native boars for 96 h of liquid preservation at 17℃.

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of ac-tin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to 100 μM of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

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-Rg1. Varying concentrations of ginsenoside-Rg1 (0, 25, 50 and 100 μM/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-Rg1 groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the 50 μM/ml ginsenoside-Rg1 group (61.0±4.65%) than in the control (46.6±7.02%), 25 μM/ml (46.2±4.76%), and 100 μM/ml ginsenoside-Rg1 (52.0± 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±0.82%) in the 50 μM/ml ginsenoside-Rg1 group than in the other groups. Here, we report that ginsenoside-Rg1 affects the motility and viability of boar spermatozoa. Moreover, ginsenoside- Rg1 can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.

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.

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY (; EG and ; 7% G) than 8% LDL (; EG and ;G). Treatment of 4% LDL + 5% EY-EG () has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG () among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

Endocrine disruptors bind to hormone receptors on sperm membrane, therefore spermatozoa are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of this study was to compare the effects of two xenoestrogenic compounds [genistein (Gen) and 4-tert-octylphenol (OP)] to those of two steroids [estrogen (E2) and progesterone (P4)] on boar sperm % motility and motion kinematics of in vitro. Porcine spermatozoa were incubated with various concentrations (0.001～100 μM) of each chemical for 15 or 30 min, and then assessed % motility and sperm motion kinematics using computer assisted sperm analyzer (CASA). Each chemical decreased sperm % motility, and OP decreased VSL and VAP compared with untreated control(p<0.05). E2 stimulated the motion kinematic changes except VCL. Moreover, Gen had effects on VCL and VAP alterations after 30 min incubation. In summary, since all chemicals studied effectively altered sperm % motility and motion kinematics, it was concluded that porcine spermatozoa could be a useful model for in vitro screening of potential endocrine disruptors.

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 ( vs , 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 ( vs , p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ( vs , 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.

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.

The objective of this study was two folds: to investigate the relationship between paternal identification rate and sperm quality parameters such as motility and sperm chromatin structure assay after heterospermic insemination; to see if mutual complement between tests and development of useful technique to enhance the fertility in artificial insemination. In individual boar's fertilizing ability, 3 high fertility boars showed significantly high fertility (p<0.05) compared to 3 low fertility boars, but there was no difference in litter size between two groups. Sperm motility test in pooled and individual semen using computer assisted sperm analysis (CASA) revealed that no significant difference among boars. The high fertile boar showed tendency of low %Red (High red fluorescence/green+red fluorescence) in sperm chromatin structure assay (SCSA) but paternal identification rate from piglets did not differ after heterospermic insemination. The correlation coefficient between individual or pooled semen function test and farrowing rates were well correlated as follows: %Red with litter size (r= - 0.53, p=0.03); %Red with paternal identification rates (r=-0.51, p=0.03); paternal identification rates with litter size (r=0.57, p=0.02). These results indicate that sperm chromatin structure assay and sperm quality parameter test in pooled semen are useful method to predict and evaluate the fertilizing capacity after heterospermic insemination in boars.

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37/with hFF, Group II: 32/withGroup III: 37/without, Group IV:32 /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.91.9%. After 24 hours culture, sperm motility was 23.52.1% (Group I), 8.11.1% (Group II), 10.4 1.4% (Group III) and 4.00.8% (Group IV), respectively. After 48 hours, the motility had been changed as 322.3% (Group I), 14.31.7% (Group II), 5.3 1.4% (Group III) and 4.30.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37 was higher than those of group II at 32. But, sperm viability of group I cultured at 37 was lower than those of group II at 32 (54.44.1% vs. 59.43.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.