• Journal of Embryo Transfer
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• v.14 no.1
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• pp.31-37
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• 1999

The objective of this study was to optimize the selection of sperm sources, optimal culture systems and vitrification method depends on sperm sources. The oocytes were inseminated with either KPN 105, 114, 191, SNU 101, 102, 103 or epididymis and then embryos inseminated were cultured in oviductal cell co-culture or HECM-6 as defined me dium. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The results obtained were as follows: 1. The cleavage(86.2 or 84.7%) and development rates to blastocyst (30.6 or 32.0%) were not significantly different between oviductal cell co-culture or HECM-6 culture systems(P<0.05). 2. To determine the optimal sperm sources for using IVF in this system, cleavage rates in KPN 191 and SNU 101 (74.2, 55.8%) were significantly lower rather than those in KPN 105, 114, SNU 102, 103 or epididymis (86.7, 85.1, 89.8, 85.5 or 81.2%), but development rates to blastocyst in KPN 114, SNU 103 or epididymis sperm (30.0, 33.0 or 28.6%) were significantly higher rater than those in KPN 105, 191, SNU 101, 102(21.4, 15.4, 14.9 or 25.4%), respectively (P<0.05). 3. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The survival rates were not significantly different among sperm sources (89.6%: 43/48 ; 90.1%: 46/51 ; 83.3% : 20/24). These results obtained indicate that the defined medium, HECM-6, could be use to produce of IVP bovine embryos. Since the frozen semen must be required to maintain of unvariation data in IVP embryo production system, KPN 114 and SNU 103 produced in our laboratory were useful for this purpose. The blastocysts produced by different sperm sources as KPN, SNU or epididymis were vitrified by OPP vitrification method and survived very high rates. The OPP vitrification method could be susceptibility to use of IVP bovine blastocyst embryos.

• Journal of Animal Science and Technology
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• v.63 no.5
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• pp.1194-1203
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• 2021

Preparation of recipient stallions is critical step to produce donor spermatogonial stem cell (SSC) derived sperm using transplantation technique. This study was conducted to evaluate the effects of intravenous busulfan infusion on germ cell depletion, semen production, and libido in stallions. Six Thoroughbred stallions were separated into two treatment groups: 1) a multiple low-dose (2.5 mg/kg bw for the first 4 weeks and 5 mg/kg bw for the 5th week); and 2) control group treated with PBS. Testicular samples were obtained at 11 weeks and classified into three different patterns of spermatogenesis, such as normal, Sertoli cell only, and destroyed. Semen collection and libido experiments were performed 1 week before treatment, and 4 and 8 weeks after treatment. For the sperm analysis, total spermatozoa and motility were measured using a light microscope with a motility analyzing system. In the multiple low-dose group, the numbers of tubules categorized as Sertoli cell only were significantly higher than those in the control as well as the total population and total/progressive motility of sperm were significantly decreased 8 weeks after the start of the treatment. The sperm production and motility in the multiple low-dose group appears to be reduced, while libido was maintained. In conclusion, multiple administration of 2.5 mg/kg bw busulfan depletes endogenous germ cells in the stallion recipients for SSC transplantation.

• 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.15 no.4
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• pp.500-503
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• 2002

This study was carried out to investigate the effects of season influencing semen characteristics, frozen-thawed sperm viability and testosterone concentration in Duroc boars. There were no significant differences in the semen volume and sperm concentration of Duroc boars among spring, summer, autumn and winter. However, the pH of sperm-rich and sperm-poor fractions in autumn and winter season was higher than in spring and summer season in Duroc boars. Sperm motility and normal acrosome of raw semen in Duroc boars did not differ significantly among spring, summer, autumn and winter. However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer, autumn and winter. Serum testosterone concentrations in Duroc were higher in spring than summer, autumn and winter. In conclusion, when serum testosterone concentrations were higher in seasons, frozen-thawed sperm viability in Duroc boars were higher.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1369-1373
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• 2004

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and $10{\times}10^6$ sperm/ml than in 0.2 and $1{\times}10^6$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in $1{\times}10^6$sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at $4^{\circ}C$ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.9
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• pp.1199-1203
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• 2011

The objective of this study was to examine the binding potential of sperm to the epithelium of the sperm storage tubules (SST) in vitro and in vivo to assess the functional maturation of fowl sperm. Sperm from the testis, epididymis, as well as the proximal, middle and distal vas deferens were incubated in vitro with either the uterovaginal junction (UVJ)- or infundibular tissue containing SST at $39^{\circ}C$ for 30 min. Aliquots of sperm were also artificially inseminated into the uteri of hens, and the UVJ and infundibulum were collected 24 h post artificial insemination (AI). After incubation and AI, tissues were washed to remove loosely adhered sperm and subjected to fluorescence staining with 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) for counting the number of bound sperm per 0.25 mm2 of surface area. Sperm from the testis, epididymis, and the three segments of the vas deferens exhibited their differential (p<0.05) binding capacity, which increased gradually from the testicular to distal vas deferens sperm under both in vitro and in vivo conditions. Existing similar trend, sperm, regardless of their source had a lesser affinity to bind to the epithelium of the infundibular SST than to the UVJ-SST. These experimental results suggested that fowl sperm may undergo gradual changes in the process of functional maturation, whereby they gain the ability to bind to the epithelium of the SST during their passage through the male reproductive tract (MRT).

• Animal Bioscience
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• v.34 no.12
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• pp.1912-1920
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• 2021

Objective: This study investigated the effects of 5-aminolevulinic acid (5-ALA) on the motility parameters, mitochondrial membrane depolarization, and ATP levels in chicken sperm. Methods: The pooled semen from Barred Plymouth Rock males was used. In the first experiment, the semen was diluted 4-times with phosphate-buffered saline (PBS (-)) containing various concentrations (0, 0.01, 0.05, and 0.1 mM) of 5-ALA, and then the sperm motility parameters after incubation were evaluated by computer-assisted sperm analysis (CASA). In the second experiment, the semen was diluted 4-times with PBS (-) containing 0.05 mM 5-ALA, and then sperm mitochondrial membrane depolarization and ATP levels after 1.5 h of incubation were analyzed with the MitoPT^® JC-1 Assay and ATP Assay kits, respectively. In the third experiment, the semen was removed from the seminal plasma and resuspended with the mediums of PBS (-), PBS (-) supplemented with CaCl₂ and MgCl₂ (PBS (+)) + 5-ALA, PBS (+) + caffeine, and PBS (+) + caffeine + 5-ALA. Then, the sperm motility parameters after incubation were evaluated by CASA. In the last experiment, the semen was treated with the mediums of PBS (-), PBS (-) + 5-ALA, 5.7% glucose, 5.7% glucose + 5-ALA after removing the seminal plasma, and then the sperm motility parameters were evaluated by CASA. Results: The addition of 0.05 mM 5-ALA significantly increased the chicken sperm motility, progressive motility, linearity, average path velocity, curvilinear velocity, straight-line velocity, and the wobble. The sperm mitochondrial membrane depolarization was also increased by the 5-ALA treatment. The 5-ALA treatment decreased the sperm ATP levels. Both the caffeine treatment and glucose treatment decreased the sperm motility during incubation period. Conclusion: 5-ALA might increase sperm mitochondrial membrane depolarization to utilize the ATP for enhancing sperm movement.

• Animal Bioscience
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• v.35 no.2
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• pp.166-176
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• 2022

Objective: Sperm is particularly susceptible to reactive oxygen species (ROS) stress. Glutathione (GSH) is an endogenous antioxidant that regulates sperm redox homeostasis. However, it is not clear whether boar sperm could utilize cysteine for synthesis GSH to protect sperm quality from ROS damage. Therefore, the present study was undertaken to elucidate the mechanism of how cysteine is involved in protecting boar sperm quality during liquid storage. Methods: Sperm motility, membrane integrity, lipid peroxidation, 4-hydroxyIlonenal (4-HNE) modifications, mitochondrial membrane potential, as well as the levels of ROS, GSH, and, ATP were evaluated. Moreover, the enzymes (GCLC: glutamate cysteine ligase; GSS: glutathione synthetase) that are involved in glutathione synthesis from cysteine precursor were detected by western blotting. Results: Compared to the control, addition of 1.25 mM cysteine to the liquid storage significantly increased boar sperm progressive motility, straight-line velocity, curvilinear velocity, beat-cross frequency, membrane integrity, mitochondrial membrane potential, ATP level, acrosome integrity, activities of superoxide dismutase and catalase, and GSH level, while reducing the ROS level, lipid peroxidation and 4-HNE modifications. It was also observed that the GCLC and GSS were expressed in boar sperm. Interestingly, when we used menadione to induce sperm with ROS stress, the menadione associated damages were observed to be reduced by the cysteine supplementation. Moreover, compared to the cysteine treatment, the γ-glutamylcysteine synthetase (γ-GCS) activity, GSH level, mitochondrial membrane potential, ATP level, membrane integrity and progressive motility in boar sperm were decreased by supplementing with an inhibitor of GSH synthesis, buthionine sulfoximine. Conclusion: These data suggest that boar sperm could biosynthesize the GSH from cysteine in vitro. Therefore, during storage, addition of cysteine improves boar sperm quality via enhancing the GSH synthesis to resist ROS stress.

• Korean Journal of Agricultural Science
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• v.50 no.4
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• pp.941-952
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• 2023

The metabolites of agrichemicals, such as organophosphorus pesticides, are known to be more hazardous than their parent pesticides. 3,5,6-trichloro-2-pyridinol (TCP) is a major degradation product of chlorpyrifos, one of the organophosphate insecticides widely used in agriculture. In vivo or in vitro exposure to chlorpyrifos has been known to interfere with male reproductive functions, leading to reduced fertility in mammals. Therefore, this study was performed to examine the changes in the fertilization competence of boar spermatozoa exposed to TCP. Sperm samples were subjected to varying concentrations of TCP (10, 50, 100, 200 µM) and different periods of incubation. Sperm motility, motion kinematics, viability, acrosome integrity, intracellular reactive oxygen species (ROS) production, and gene expression levels (ODf2, ZPBP2, AKAP3 and AKAP4) were evaluated after exposure of the sperm to TCP. A significant dose-dependent reduction in motility was observed in sperm samples incubated with TCP compared to the controls after both incubation periods. Sperm viability was significantly decreased in samples incubated with 50, 100, and 200 µM TCP in both incubation periods. A significantly lower percentage of normal acrosomes and gene expression levels were observed in sperm samples exposed to 50, 100, and 200 µM TCP after both incubation periods, compared to the controls. There was a significant increase in the ROS production in spermatozoa incubated with 100 - 200 µM TCP after both incubation periods. Consequently, the direct exposure of boar spermatozoa to TCP interferes with sperm functions and leads to decreased fertilization. In order to identify and address the various causes of reproductive decline, the impact of chemical metabolites needs to be discussed in depth.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.5
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• pp.644-650
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• 2023

We examined the effects of serotonin on the spawning response, sperm motility, and D-shaped larva production in Eastern Gooeyduck clam Panopea sp. based on the sperm densities at fertilization and washing after mixing the eggs and sperm. The highest spawning induction was found showed in females and males injected with 1 mL of 2 mM serotonin. The spawning responses in females and males were higher at concentrations greater than 1 mM and 0.75 mM, respectively. Regarding the activities of sperm in sea water after serotonin injection, the sperm showed activity at >90.0% until 120 mins. We also examined the effects of sperm concentration at the fertilization and washing times after mixing the eggs and sperm. We confirmed that washing within 1 minute at a concentration of 1,500 sperms/mL or less can prevent egg destruction by polyspermy and secure a large number of D-phase larvae. These results should be useful for developing the aquaculture process for Eastern Gooeyduck clam, Panopea sp.