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.

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, 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. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

In this experiment, we determined the effect of curcumin supplementation in freezing buffer for miniature pig sperm cryopreservation. Each ejaculate was diluted with modified Modena B extender and mixed with lactose-egg yolk (LEY extender, 80% v/v lactose solution [310 mM], 20% v/v egg yolk, and100 μg/mL kanamycin sulfate) and LEY-glycerol Orvus ES Paste (LEYGO, 89.5% v/v LEY, 5% v/v glycerol, 1.5% v/v Orvus ES Paste), 100 mM trehalose supplemented with 0, 10, 50, 100, and 500 μM of curcumin from turmeric, respectively. Following equilibration, the 0.5 mL French straws were frozen and plunged into LN2 tank for 7 days at least. Sperm parameter and oxidative byproducts were determined by the computer assisted sperm motility analysis (CASA) and fluorescence-activated cell sorting (FACS) as compared with each groups.Supplementation of curcumin had no effect on sperm motility, progressive motility and curvilinear velocity. However, average-path velocity and straight-line velocity were significantly higher in 10 μM curcumin group (100.9±8.8 μm/s, 61.7±2.9 μm/s, respectively) than control group (77.8±3.9 μm/s, 46.4±3.0 μm/s, respectively) (p < 0.05). In addition, the level of the O2 radical and H2O2 were comparatively decreased in curcumin groups by evaluation of ethidium and DCF fluorescence. According to the results, curcumin can improve sperm kinetic variables and alleviate ROS induced cryoinjury to pig 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.

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus (GalT-MCP/-MCP) as well as expressing MCP at GalT gene loci with CD73 expression (GalT-MCP/+/CD73). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, GalT-MCP/+/CD73, and GalT-MCP/-MCP pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

The objective of this study was to investigate the influence of the addition of caffeine (alkaloid family) to the ejaculates of boar sperm quality as well as investigate their optimum concentrations for increasing the movement of sperms. Semen was collected from 9 boars by the gloved-hand technique one week interval. Semen followed by cryopreservation with egg yolk extender freezing medium using freezing protocol. The collected semen were frozen on the same day. Motility was assessed for % motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). Frozen boar sperms were thawed in Beltsville Thawing Solution (BTS) with 5, 10, and 15mM caffeine were then incubated at 38 celsius degree for 20 minutes. In experiment 1, semen were diluted BTS and addition of different concentration of caffeine to the pre-freeze semen cryopreservation. In experiment 2, incubation of frozen-thawed sperm in BTS supplemented with different concentration of caffeine for 20 minutes improved (P<0.05) after semen cryopreservation-thawing on sperm quality. After thawing significantly increased progressive and total motility. The addition of 10 mM caffeine to cryopreserved semen after thawing significantly increased progressive and total motility compared with other treatment. These result suggest that caffeine enhanced post-thaw motility of cryopreserved boar sperm when added after thawing.

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.

Post-ejaculation of sperms into the female reproductive tract, acquisition of sperm capacitation is an essential step in the fertilization process. Accordingly, during in-vitro fertilization, the successful fertilization requires necessarily induction of capacitation in the retrieved sperms. To date, many candidate substances have been considered as capacitation inducers. However, there were no reports about the comparison of efficiency inducing sperm capacitation among diverse capacitation inducers. Therefore, we tried to determine an inducer showing the best capacitation performance in mouse sperms by comparing the preimplantation development of in-vitro-fertilized embryos using sperms experiencing capacitation by a variety of capacitation inducers. For these, calcium, progesterone, bovine serum albumin (BSA), heparin, lysophosphaticylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentration of each inducer were determined by accessing ratio of sperms experiencing acrosome reaction using coomassie G-250 blue staining. Subsequently, in vitro fertilization was performed using sperms incubated in each optimized concentration inducer. The ratio of fertilized oocytes was observed. As the results, Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 μM progesterone, 50 mM heparin, and 100 μM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization. From these results, we could identify that, among diverse sperm capacitation inducers, 2.7mM calcium and 0.3% (w/v) BSA were the most effective inducers for in vitro fertilization.

The purpose of this study is to evaluate the effects of astaxanthin added to freezing buffer on semen parameters, total sperm oxidation stress after post-thawing of boar sperm and lipid peroxidation (LPO) which is caused by reactive oxygen species (ROS) in sperm membrane. Varying concentrations of astaxanthin (0, 10, 50, 100 and 500 μM) were used in the freezing buffer during cryopreservation to protect the DNA of thawed miniature pig sperm. Semen parameter was measured using computer assisted sperm analysis (CASA) for sperm motility and determine ROS rate, oxidative stress of boar sperm using fluorescence-activated cell sorting (FACS). Sperm motility was higher (p<0.05) in the astaxanthin group than in the control group. Sperm motility and the number of progressive motile sperm was higher (p<0.05) in the astaxanthin 500 μM group (66±1.7%) than in the control group (49.8±4%). In ROS evaluation, the astaxanthin group lowered intracellular O2 and H2O2 in viable sperm. The Yo-Pro-I/HE and PI/H2DCFDA staining as revealed using flow cytometry was lower in astaxanthin groups than in the other groups. As the result, we found that astaxanthin could protect the sperm plasma membrane from free radical and LPO during boar sperm post-thawing.

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.

본 연구의 목적은 능성어와 자바리의 정자동결을 위한 간편한 실험법개발이다. 희석제와 동해 방지제가 정자동결에 미치는 효과를 파악하고자 운동성성과 생존율을 조사하였다. 동결실험에 서 300 mM glucose와 15% dimethylsulfoxide (DMSO)를 희석제와 동해방지제로 각각 사용하였 다. 동결실험결과, 능성어 정자는 동결 5개월 후 60% 이상의 운동성을 보였고, 자바리 정자는 동결 5개월후 90% 이상의 운동성을 보였다.

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 cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.