In this study, an experiment was conducted in order to determine what cryopreservatives (CPVs) were more effective in supporting the motility and viability of sperm from experimental animals. The sperm of mice, rats, beagle dogs, and rabbits were frozen using different CPVs, including DMSO, TYB, and Sperm CryoProtec. The results from freezing the sperm of each laboratory animal in Sperm CryoProtec showed a high level of sperm motility and viability in sperm samples from mice, rats, and beagle dogs melted at the end of the first week. For rabbits, a high level of motility was observed in sperm thawed during the first week, whereas a high level of viability was observed in sperm thawed during the second week. The results of analysis of sperm motility and viability using different CPVs according to laboratory animals showed a significantly higher level of sperm motility (26.28%) and viability (36.20%) for mice in Sperm CryoProtec and the lowest levels of motility and viability were observed in DMSO (p < 0.05). Significantly higher levels of motility (27.94%) and viability (37.94%) were observed for rats in Sperm CryoProtec compared with TYB, which showed the lowest levels of motility and viability (p < 0.05). The study findings described above suggest that the selection of appropriate cryopreservatives is required for each experimental animal. This is because there are differences in the levels of sperm motility and viability of experimental animals depending on the CPVs that are typically used for freezing human sperm, including Sperm CryoProtec and TYB.

Subgroups of sperm which share similar motility features documented in mammals indicate between-subject variations that might be related to fertilizing potential of the respective ejaculates. The objectives of this study were to define subpopulations of motile sperm in Gyr falcon semen using kinematic parameters driven by Computer Assisted Semen Analysis (CASA) and to investigate the subjectrelated variations in these subpopulations. A total of 24 fresh ejaculates from 6 falcons were used to assign each of the 20473 sperms into 3 subpopulations by a multivariate cluster analysis. The proportion of sperms in different sub-populations were compared among subjects by a generalized linear model and repeatability of sperm frequency in different subpopulations was investigated by corelation analysis. The resulting 3 categories of sperm indicated significant differences in all kinematic parameters (p < 0.05). Subpopulation 1 (15.91%) contained sperms with the highest velocity and progressiveness of movement trajectory while subpopulation 3 (6.4%) included the least progressively motile sperms. Proportion of rapid and medium progressive sperm were consistently higher in the ejaculate of three falcons compared to the two other birds which also had the highest proportion of slow non-progressive sperms (p < 0.05). Respective proportion of sperms in each subpopulations indicated significant repeatability over multiple measurements (p < 0.05). In conclusion, subpopulations of motile sperm in Gyr falcon can be identified using kinematic parameters generated by CASA. Individual differences in the proportion of these subpopulations might have potential application for identifying the males with higher fertilizing capacity.

In this study, we examined number, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in bull. Six testicles with epididymides were castrated from six bulls (mean±standard error, age of days = 441.3±9.6, body weight (kg) = 367±8.4, scrotal circumference (cm) = 30.7±0.4) at Hanwoo Research Institute, NIAS and transported to laboratory within 1 hour. Testicular weight, length, width and circumference were recorded. Epididymis in each bull was randomly used for recovery of spermatozoa. Epididymis was divided into six regions: efferent duct (ED), caput, corpus, proximal cauda (Pcauda), distal cauda (Dcauda) and vas deferens (VD). In experiment 1, we examined sperm number of each region of epididymis. Each region of epididymis contained different number of spermatozoa: ED (37.8±15.7 × 106cells/ml, 8.2%), caput (93.6±18.8 × 106cells/ml, 20.2%), corpus (33.0±8.5 × 106cells/ml, 7.1%), Pcauda (104.2±23.5 × 106cells/ml, 22.5%), Dcauda (180.5±32.5 × 106cells/ml, 39.0%) and VD (14.0±5.0 × 106cells/ml, 3.0%). In experiment 2, sperm motility of each epididymal region was examined by computer assisted sperm analysis (SCA, MicroOptic) system. Sperm motility was divided into 4 groups (fast progressive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentages of fast progressive of Pcauda and Dcauda (11.0±2.3 and 15.4±3.6%) were significantly higher than that of ED, Caput, Corpus and VD which is 0.1±0.1, 1.5±0.6, 1.9±0.7 and 0.3±0.2%, respectively (p<0.05). In experiment 3, percentage of intact plasma membrane spermatozoa of each regions were examined by hypoosmotic swelling test. Percentages of intact plasma spermatozoa were not significantly different among six regions of epididymis: ED, caput, corpus, Pcauda, Dcauda and VD which is 68.0±8.6, 74.0±5.3, 68.5±6.2, 70.8±5.5, 71.0±5.8 and 64.6±10.8%, respectively. In conclusion, in the present study, we found out distribution, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in Hanwoo bull. These results will be contributed to basic research about spermatozoa transportation and characters in epididymis of bull.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm 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 used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 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 ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

During the freezing and thawing process, fatty acids in the plasma membrane of sperm are released, which results in a functional damage of sperm. Sperm with functional loss due to cryo-damage result in a decrease in fertility. Previous studies have shown that the addition of one of the fatty acid alpha-linolenic (ALA) with carrier proteins improves the stability of plasma membrane and reduces the damage. In this experiment, we focused on the functional aspects of the plasma membrane of sperm and experimented with motility and morphology. For preparation of ALA-carrier protein complex, 3 ng/ml ALA was mixed with 0.7 μg/ml bovine serum albumin (BSA) or 14 ng/ml methyl-β-cyclodextrin (MBCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in 20% egg yolk freezing extender containing ALA, BSA, MBCD, ALA+BSA, ALA+MBCD. The frozen boar sperm was thawed at 37.5 ℃ for 45 sec in water-bath. The sperm motility and morphological abnormalities were evaluated under a phase-contrast microscope at 200 × magnification and randomly counts of 200 sperm each sample. In results, motility of frozen-thawed sperm was increased in all treatment groups. In particular, there has been significant improvement in ALA+BSA and ALA+MBCD treatment groups than control (p<0.05). However, there was no significant difference in ALA, BSA and MBCD treatment groups. Morphological normalities in frozen-thawed sperm was reduced in complex treatment groups (p<0.05). However, there was no significant difference in single treatment groups. In both motility and morphology characteristics, ALA+BSA and ALA+MBCD treatment group was higher than all treatment groups. In conclusion, the addition of ALA with carrier proteins during cryopreservation has a positive effect in its functional aspect. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

This study was to investigate effect of tunicamycin (TM) on sperm viability, mitochondrial activity and motility in boar semen. Collected sperm were incubated with semen extender containing 0, 1, 2, and 5 μM TM for 3, 6 and 9 h. Sperm viability was analyzed using SYBR14/PI doubling staining, and mitochondrial activity was detected using Rhodamine123/PI staining methods. Sperm viability, mitochondrial activity and motility were measured every 3 h during incubation. In results, boar sperm viability, mitochondrial activity and motility were significantly decreased in 2 and 5 μM TM groups compare to control group at all incubation time (p<0.05). In addition, mitochondrial activity and motility were significantly decreased in 1, 2, and 5 μM TM groups compare to control group at 9 h after incubation (p<0.05). These results suggest that TM can inhibit sperm viability, mitochondrial activity and motility in boar semen, and it may influence on the fertility of sperm.

Antioxidants have been added to cryoprotectant or in vitro culture medium for sperm to reduce the detrimental damage, such as reactive oxygen species. However, curcumin, an antioxidant, shows dual effect on the viability and progressive motility of bovine sperm exposed to hydrogen peroxide. Low concentration of curcumin increases sperm viability and progressive motility, whereas high concentration of curcumin reduces them. This study was performed to identify whether TREK-1 channel is related to low sperm viability and motility induced by high concentration of curcumin. Curcumin reduced TREK-1 channel activity in a dose-dependent manner. TREK-1 channel was expressed in sperm obtained from Korean native bull. Treatment with TREK-1 channel blockers, such as curcumin, fluoxetine, GdCl3, and spadin, significantly reduced sperm viability and motility (p < 0.05). However, TREK-1 channel activators showed different effect; linoleic acid showed an increase in sperm viability and motility, and wogonin did not affect them. These results show that TREK-1 channel is involved in the regulation of sperm viability and motility. In particular, high concentration of curcumin might reduce sperm viability and progressive motility of Korean native bull through blockage of TREK-1 channel.

The present study was conducted to investigate the effect of BHT supplementation on sperm motility, viability, acrosomal integrity and plasma membrane integrity after frozen-thawing. One ejaculate was collected from one fertile Hanwoo bull by using artificial vagina at Hanwoo Research Institute. The ejaculate was transferred to laboratory immediately and diluted with pre-warmed semen extender (Optixcell, France) (1:1). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and divided into 5 groups according to BHT concentration (0, 0.5, 1.0, 2.0 and 4.0 mM) and cryopreserved in LN2 tank until evaluation. Frozen-thawed semen was transferred to 1.5 ml tube and incubated for 0, 2 and 4h. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain). There were not significant effects of BHT supplementation (0, 0.5, 1.0 and 2.0 mM) on total motile and VSL with 25μm≥ at 0, 2 and 4h. However, 4.0 mM of BHT supplementation showed negative effect on total motile (26.3%), VSL with 25μm≥ (1.3%) at 0 h (p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method and divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). There were no significant differences of LIA, LDA, DIA and DDA on various BHT concentrations at 0 and 2 h. However, 4.0 mM BHT supplementation showed decreased LIA compared with 0, 0.5, 1.0 and 2.0 mM BHT at 4 h (34.6, 37.1, 43.6, 45.4 and 14.7% vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.01). Addition of 4.0 mM of BHT showed negative effect on plasma membrane integrity compared with that of 0, 0.5, 1.0 and 2.0 BHT at 2 h (71.9, 64.2, 64.6, 67.5 and 31.7 % vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.05). In conclusion, various BHT concentrations on optixcell extender showed no improvement on sperm motility, viability and plasma membrane integrity.

In the present study, we evaluated the effect of glucose-fructose and sucrose supplementation in glycerol-free tris (GFT) on sperm motility, viability, ROS level, apoptosis (BAX and BCL2) and motility (SMCP) related gene expression of dog sperm according to different post-thaw incubation time. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. The progressive motility, viability, ROS (H2O2) level and mRNA expression of spermatozoa were evaluated according to post-thaw incubation time (0 h, 3 h and 6 h) at 24℃. ROS was assessed using H2DCFDA stain by flow cytometry. The relative abundances of BAX, BCL2 and SMCP were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The motility of spermatozoa cryopreserved in GF-GFT was increased throughout the post-thaw incubation time. The motility of spermatozoa cryopreserved in S-GFT was increased at 3 h of post-thaw incubation. Whereas, the sperm ROS level in GF-GFT group was decreased at 6 h of post-thaw incubation. However, the ROS level in the group S-GFT was gradually increased with the progress of post-thaw incubation period. The post-thaw incubation had no substantial effect on mRNA expression of BAX, BCL2 and SMCP genes of dog spermatozoa in both the GF-GFT and S-GFT groups. These results indicate that GF supplementation in GFT improves the progressive sperm motility during the 6 h of post-thaw incubation with maintaining similar sperm viability and is more efficient in reducing ROS after 3 h of post-thaw incubation. The addition of GF in GFT for the cryopreservation of dog spermatozoa and post-thaw incubation would open an option to achieve more functioning spermatozoa for future assisted reproduction practices.

The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.

The present study was conducted to investigate the effect of different heights from liquid nitrogen (LN2) vapor on sperm motility and morphology after frozen-thawing. Two ejaculates were collected from 2 fertile Hanwoo bulls (A and B) by using artificial vagina at Hanwoo Research Institute. After collection, ejaculates were transferred to laboratory immediately and diluted with semen extender (Optixcell, France). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and cooled at 4°C for 4 h and loaded to 0.5 ml straws. The straws were divided into 2 groups. Straws were placed in 3 or 9 cm of LN2 vapor for 14 min and then plunged into LN2 tank and cryopreserved until evaluation. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain) after frozen-thawed. In bull A, 3cm group showed higher percentages of total motility, VSL with 25μm and VAP compared those with 9cm group (98.0 vs. 93.4%, 62.4 vs. 54.0% and 98.6 vs. 93.2%, 3 vs. 9 cm, irrespectively; p<0.001). In bull B, frozen-thawed sperm of 3cm group showed higher percentages of VSL with 25μm, VCL, VSL, VAP and BCF compared with those of 9cm group (43.5 vs. 26.0%, 123.8 vs. 111.6 μm, 62.9 vs. 57.3 μm and 81.5 vs. 72.5 μm; 3 vs. 9 cm, irrespectively; p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). In bull A, frozen-thawed sperm of 3 and 9cm groups showed no significant difference in LIA, LDA, DIA and DDA. In bull B, 3 cm group showed higher LIA and lower DIA compared with those of 9 cm group (73.2 vs. 23.7% and 23.7 vs. 32.2%, 3 vs. 9 cm, irrespectively; p<0.001). We suspected that 3 cm vapor on LN2 vapor might be affected positively spermatozoa viability and acrosomal integrity compared with 9 cm group. In conclusion, semen freezing procedure in the present study will improve sperm quality after frozen thawing.

The present study was aimed to determine the effect of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on sperm motility, viability and reactive oxygen species (ROS) level. Experimental groups were allocated into Lactose-egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/L GTE in LEY) and β-ME (50 μM β-ME in LEY). Spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM (final sperm concentration: 1 × 108/mL). Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. Following thawing at 37°C for 25 sec, sperm viability and ROS level were measured using fluorescent double stain Fertility® and cytometry, respectively. Motility and viability of GTE supplemented-group were higher than those of control and β-ME without significance. ROS level in GTE group showed significantly lower than control (P < 0.05). In conclusion, GTE supplementation in boar sperm freezing extender can reduce ROS generation during freezing.

The present study was conducted to investigate the effect of caffeine and theophilline on sperm motility during in vitro fertilization (IVF). Briefly, commercial boar semen was centrifuged and resuspended (5x107 sperms/ml) with fertilization medium (mTBM) supplemented with 2 mM caffeine (Caf), 5 mM theophylline (The), 2 mM caffeine + 5mM theophylline (Caf + The), and not supplemented control. The semen parameters of the four groups were analyzed by computer-assisted semen analysis (CASA, Medical Supply Co. Ltd) system at 6 h as time for IVF at 38.5 C under 5 % CO2 in air. The groups were examined 11 semen parameters such as total motility (TM), curvilinier velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), and hyperactivated (HYP), etc. A total motility of control group (41.3 %) at 6 h showed significantly (P<0.05) higher than those of other groups (Caf, 37.2; The, 35.2; Caf + The, 30.1 %). Results from many other sperm parameters indicated that the theophylline and caffeine negatively affected on sperm motility during IVF. These results suggested that the supplementation of caffeine and/or theophylline was not essential for IVF in pigs. To prove this suggestion, further studies are needed to analyze fertility and embryonic development after IVF with or without the supplementation of caffeine and/or theophylline.

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. Phospholipase C zeta (PLCz) is important enzyme in spermatogenesis, but their effect has not been confirmed in pigs yet. Therefore, this study was aimed to analyze their association with sperm motility and kinematic characteristics. DNA samples from 124 Duroc pigs with records of sperm 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 subjected. A SNP in non-coding region of PLCz g.158 A > C was associated with MOT (p < 0.05), VCL (p < 0.01), LIN (p < 0.01) and ALH (p < 0.05) in Duroc population. Therefore, we suggest that the intron region of the porcine PLCz gene may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

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 candidate gene for 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), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] 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.

Although cryopreservation of sperm is routinely used for clinical requirement, it has some problems, such as high generation of reactive oxygen species (ROS) and cold-shock. To reduce the detrimental damage in sperm, anti-oxidants were added to cryoprotectant for sperm. Curcumin is one of anti-oxidants, which are added in cryoprotectants. However, recent studies have demonstrated that curcumin decreases sperm viability and motility. This study was performed to identify the effect of curcumin on hydrogen peroxide (H2O2)-exposed bovine sperm, which were cryopreserved-thawed. In H2O2-exposed bovine sperm, reactive oxygen species (ROS) were significantly reduced by treatment with curcumin in a dose-dependent manner (p < 0.05). Among tested concentrations of curcumin (1 to 50 μM), 30 and 50 μM curcumin showed anti-oxidant effect on H2O2-induced ROS generation. On the other hand, combination of 30 or 50 μM curcumin with anti-oxidant H2O2 increased the percentage of apoptotic sperm compared to only H2O2 treatment. Sperm viability was also decreased in the combination of 30 or 50 μM curcumin with H2O2 as judged by FDA/PI staining. H2O2–induced decrease in sperm progressive motility was recovered by treatment with 1 μM curcumin. These results show that high concentration of curcumin has anti-oxidant effect, but it has also cytotoxic effect on bovine sperm. Sperm viability and motility might be more affected by cytotoxic signals of curcumin compared to antioxidant signals.