• Journal of Embryo Transfer
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• v.31 no.4
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• pp.355-365
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• 2016

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 $1{\mu}g/ml$) 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 $1{\mu}g/ml$ (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 $0.01{\mu}g/ml$ AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and $0.1{\mu}g/ml$ AFP (p<0.05), but increased with $1{\mu}g/ml$ AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.100-105
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• 2019

The purpose of this study was to evaluate the effect of addition of ethylene glycol, glycerol and sucrose to TCG (Tris, Citric Acid, Glucose, Egg Yolk) and DMSO Frozen. The extender containing Egg yolk concentration (10%, 20%) affects viability and acrosome morphology of rabbit sperm. Sperm viability was then assessed for the freezing extenders TCGD (Tris + Citricacid + Glucose + DMSO), TCGED (Tris + Citricacid + Glucose + Egg yolk + DMSO), TCGGD (Tris + Citricacid + Glucose + Glycerol + DMSO) and TCGSD Tris + Citricacid + Glucose + Sucrose + DMSO) during thawing at 38℃. for 20 seconds, respectively. TCG + 10% egg yolk (viability: 77.0 ± 0.8, NAI: 73.3 ± 0.9) was significantly (sperm viability and normal acrosome interaction (NAI)) higher than TCG + 20% egg yolk (70.7 ± 1.1, 70.0 ± 0.9) in the sperm normalcy analysis according to the yolk concentration. TCGGD (53.4 ± 0.1, 62.3 ± 0.4), TCGSD (61.3 ± 0.0, 67.1 ± 0.1) sperm viability and normal acrosome interaction (NAI) in frozen spermatozoa are TCGD (46.4 ± 2.8 and 56.3 ± 1. 4) and TCGED (23.0 ± 1.1 and 54.6 ± 1.4) extenders was thawed at 38℃ for 20 seconds. According to the results from each frozen bulking agent, sperm membrane integrity by hypotonic swelling test (HOST) analysis in TCGGD (59.8 ± 0.7), TCGSD (59.3 ± 0.5) was significantly high compared to other experimental groups (p < 0.05). In conclusion, these results suggested that TCGGD and TCGSD extenders enhance survivability of rabbit sperm after frozen-thawing.

• Reproductive and Developmental Biology
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• v.40 no.4
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• pp.33-37
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• 2016

This study was conducted to evaluate effect of ${\alpha}$-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 $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ 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.

• Journal of Aquaculture
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• v.11 no.1
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• pp.67-75
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• 1998

Experiments were performed to obtain cryopreservation techniques of black seabream (Acanthopagrus schlegeli) sperm. For sperm collection, brood stock reared in recirculating seawater system and fed with the commercial feed during experimental period. The results indicated that following cryopreservation method in block seabream sperm could be employed. Post-thaw survival rate of sperm revealed the highest value ($80{\pm}1.4$%) in 3% sodium citrate as a diluent for the cryopreservation. Cryopreserved sperm diluted with 5.4% glucose showed the highest fertilization rate to the ovulated eggs. Glycerol was a better cryoprotectant than dimethyl sulfoxide in sperm cryopreservation : survival rate and fertilizing capacity of cryopreserved sperm were decreased according to increase of glycerol concentration and varied in renges of 0.8~59.3% and 32.5~69.4% with 5~30% glycerol, respectively. A few of cryopreserved spermatozoa showed the enlarged head with granulated chromatin and ruptured plasma membrane by freezing and thawing injuries compared with unfrozen normal spermatozoa.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.98-99
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• 2006

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.10a
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• pp.158-158
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• 2003

Bioassays using gametes of sea urchins are widely used in ecotoxicological assessments of marine environments. Since most of sea urchin species in Korean coastal water spawn from spring to autumn, bioassay with them during the winter is impossible. In the course of developing standard methods for bioassays with Korean species, we found a winter-spawning starfish, Asterias amurensis, Since reproductive mode of asteroids is similar to echinoids, the bioassay protocol for sea urchins could be applied similarly to the starfish. Here, we tested and determined several conditions for the acceptability of bioassay with A. amurensis. The least required time for formation of fertilization membrane of fertilized eggs to be easily distinguished from unfertilized ones was 60 min. The threshold of sperm to egg ratio that could make acceptable fertilization rates in controls was 3000. The allowed time for manipulation of sperm after dilution in seawater was at most 3 hr. The optimal exposure time of sperms when the response against toxicant solution was relatively stable was in the range of 20-60 min. The tolerance range of sperms to the salinity of test solution was 26-38 psu. The sensitivity of A. amurensis sperm was intermediate among marine organisms commonly used in aquatic toxicity tests. The sperm bioassay with A. amurensis can be satisfactorily applied to toxicity assessments of marine environments.

• Korean Journal of Agricultural Science
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• v.49 no.2
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• pp.259-267
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• 2022

Although pesticides are recognized as necessary substances to improve agricultural production, exposure to pesticides is known to have a direct or indirect adverse effect on the reproductive function of mammals. The present study examines the effects of mancozeb, a well-known fungicide, on the fertility capacity of spermatozoa. Boar spermatozoa exposed to varying concentrations of mancozeb (0.01 - 0.5 µM) were evaluated for motility, motion kinetic parameters, viability, acrosome integrity and the generation of intracellular reactive oxygen species (ROS) after 30 min or 2 hrs of incubation. A significant reduction in the motility of spermatozoa was observed upon exposure to mancozeb. Similarly, there was a significant reduction of the motion kinematics of sperm treated with mancozeb as compared to untreated controls (p < 0.05). The sperm viability percentage and acrosome integrity also showed dose-dependent decreases upon exposure to mancozeb. High concentrations of mancozeb (0.2 - 0.5 µM) induced higher levels of intracellular ROS production, which resulted in the loss of the sperm membrane and decreased sperm motility due to oxidative stress. Taken together, the results here indicate that direct exposure to mancozeb affects the sperm fertility capacity. Hence, careful research that examines the interaction between reproduction and environmental toxins is crucial to prevent fertility disorders in animals.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.2
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• pp.167-175
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• 2000

This paper describes the magnetic orientation of bull sperm separated into the head and the flagellum treated by DTT or heparin in a 5,400G static field. Semen samples collected from four bulls (Japanese Black) were mixed to the same sperm density. One percentage triton X-100 was used to extract the plasma membrane. The intact and demembranated sperm suspensions were treated with 20, 200, 2,000 mM DTT, 100, 1,000 or 10,000 units heparin solutions at $4^{\circ}C$ for 6 days. The decondensation of the sperm nuclei treated by DTT or heparin was examined by measuring the head area at 1, 3 and 6 days. After measuring the area, each sample was exposed to a 5,400G static magnetic field generated by Nd-Fe-B permanent magnets for 24 hours at room temperature. Results showed that the sperms were separated into the head and the flagellum through the DTT treatment. Almost of the separated heads showed that their long axis oriented perpendicularly to the magnetic lines of force, and most of the long axis perpendicularly oriented heads showed that their flat plane oriented perpendicularly in a 5,400G magnetic field. Also, the demembranation of the head tended to increase those perpendicular orientations, while those perpendicular orientations of the head declined with the decondensation of the sperm nuclei. These findings suggest that strong magnetic anisotropy for the perpendicular orientation of the long axis and the flat plane of the head occurs in the sperm nuclei in a 5,400G magnetic field. The separated flagellum showed lower parallel orientation, and the separated and demembranated flagellum showed parallel orientation to the magnetic lines of force in this magnetic field. These findings suggest that weak magnetic anisotropy of the parallel orientation of the flagellum occurs in the inside components in a 5,400G field.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.169-174
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• 2013

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of actin-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 $100{\mu}M$) 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 $100{\mu}M$ 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.