• 대한의생명과학회지
• /
• 제23권4호
• /
• pp.406-410
• /
• 2017

Curcumin is known as a natural antioxidant, decreasing oxidative stress in animal cells. Generally, oxidative stress induces reactive oxygen species in sperm and leads to decreased sperm characteristics in pigs. Therefore, this study investigated the influence of curcumin on sperm motility, viability, mitochondrial activity and plasma membrane integrity in pigs. Curcumin (0, 5 and $10{\mu}M$) was treated in boar semen, which were incubated for 9 hours in $37^{\circ}C$. Then, motility, viability, mitochondrial activity, plasma membrane integrity of sperm was analyzed every 3 hours. In the results, sperm motility was significantly increased by $5{\mu}M$ curcumin after 3 and 9 hours after incubation, and viability was significantly higher in $5{\mu}M$ curcumin treatment at 3 hours (P<0.05). Similarly, sperm mitochondrial activity and plasma membrane integrity were significantly increased by $5{\mu}M$ curcumin at 3, 6 and 9 hours after incubation (P<0.05). There results suggest that curcumin improve sperm characteristics such as motility, viability, mitochondrial activity, and plasma membrane integrity, and may exert a positive effect on sperm fertility in pigs.

• Animal Bioscience
• /
• 제35권2호
• /
• pp.166-176
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제13권6호
• /
• pp.833-840
• /
• 2003

Sperm motility of salmonid fishes is suppressed by external $K^{+}$ and initiated by decrease of $K^{+}$ concentration surrounding the sperm. It was shown that the decrease in external $K^{+}$ concentration induced not only the initiation of sperm motility, but also hyperpolarization of the plasma membrane and synthesis of cAMP in the sperm of rainbow trout, steelhead trout, and masu salmon. Inhibitors of $K^{+}$ channels, especially voltage-dependent $K^{+}$ channels, inhibited these three reactions, and the inhibitions were abolished by subsequent addition of a $K^{+}$ ionophore, valinomycin, suggesting that $K^{+}$ efflux through the $K^{+}$ channel contributes to rapid changes in the membrane potential of sperm and cAMP synthesis, thereby resulting in the initiation of sperm motility of salmonid fishes.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권3호
• /
• pp.358-363
• /
• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Reproductive and Developmental Biology
• /
• 제29권4호
• /
• pp.253-257
• /
• 2005

This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than $80\%$ of fresh sperm washed with mTLP-PVA medium at $20^{\circ}C$ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane $(36.4\~46.9\%)$ and nonfunctional mitochondrion $(55.1\~71.1\%)$ in the mTLP-PVA and BTS washing media at $20^{\circ}C$. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at $4^{\circ}C$ washing temperature than at $20^{\circ}C$ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.

• 한국동물생명공학회지
• /
• 제35권3호
• /
• pp.268-272
• /
• 2020

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSC-exo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 ㎍/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 ㎍/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

• Animal cells and systems
• /
• 제1권2호
• /
• pp.371-379
• /
• 1997

To investigate the movement of sperm head and the role of sperm neck in forward sperm motility in the Korean striped field mouse, Apodemus agrarius coreae, the morphological characteristics of the cauda epididymal spermatozoa were examined by light microscopy and scanning and transmission electron microscopy. Spermatozoa of A. agrarius coreae were characterized by the conspicuous shape of the acrosome and the long tail compared with those of other rodents. Total length of the sperm was $133\mu{m}$. The sperm head had a curved falciform shape. The head was 8.0${\mu}$m in length, and about 4.0 ${\mu}$m in width. The shape of acrosome had an openerlike form. The sperm tail (125 ${\mu}$m) consisted of four major segments: neck (0.5 ${\mu}$m), middle piece (29.5 ${\mu}$m), and principal piece plus the end piece (95 ${\mu}$m). The outer dense fibers were arranged in a horseshoe fashion, and No. 1, 5, 6, and 9 of the outer dense fibers were larger than the others. The mitochondrial bundles of middle piece were composed of a pair of arms, which surrounded the axone of the middle piece by the 45 0 angled helical structure. The total number of mitochondrial gyres was 188. In particular, the microfilament structures existed in plasma membrane of the sperm, which was adjacent to the acrosomal region on the nuclear membrane. The segmented columns were surrounded by microfilament structures, and the microfilament bundles were adjacent to the outer membrane of the first mitochondria of middle piece. This study presents for the first time the existence of microfilament structures within the plasma membrane of sperm which is located from the adjacent acrosome region to the connecting piece in sperm neck of Korean striped field mouse, Apodemus agrarius coreae. The present result suggests that the constriction and extension of microfilament in sperm neck as well as the wave-movement of sperm tail may play a role in the movement of sperm head.

• Animal Bioscience
• /
• 제34권12호
• /
• pp.1912-1920
• /
• 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.

• 한국발생생물학회지:발생과생식
• /
• 제23권1호
• /
• pp.11-19
• /
• 2019

The study was conducted to investigate the effects of alpha-linolenic acid (ALA) combined with bovine serum albumin (BSA) or methyl-beta-cyclodextrin (MBCD) on plasma and acrosomal membrane damages, mitochondrial activity, morphological abnormality, motility, and oxidative stress in frozen-thawed boar sperm. In previous our study, 3 ng/mL ALA had been shown protective effect during freezing process of boar sperm. Therefore, we used 3 ng/mL ALA in present study and ALA was combined with same molar ratio of BSA or MBCD (ALA+BSA and ALA+MBCD, respectively). To confirm the effect of two carrier proteins, same volume of BSA and MBCD without ALA were added during cryopreservation. Membrane damage, mitochondrial activity, reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were measured using flow cytometry, and movement of sperm tail as motility parameter and morphological abnormality were observed under light microscope. In results, all of sperm parameters were enhanced by ALA combined with BSA or MBCD compared to control groups (p<0.05). Mitochondrial activity, morphological abnormality, ROS and LPO levels in ALA+BSA or MBCD groups were no significant difference compared with ALA, BSA and MBCD treatment groups. On the other hand, plasma and acrosomal membrane intact, and sperm motility in ALA+MBCD group were higher than single treatment groups (p<0.05), whereas ALA+BSA did not differ. Our findings indicate that carrier proteins such as BSA and MBCD could improve the effect of ALA during cryopreservation of boar sperm, and treatment of ALA with carrier proteins enhance membrane integrity, mitochondrial activity through reduction of ROS-induced LPO.

• Clinical and Experimental Reproductive Medicine
• /
• 제49권2호
• /
• pp.110-116
• /
• 2022

Objective: Sperm vitrification leads to the production of reactive oxygen species (ROS) that can damage the functional parameters of sperm. The present study aimed to investigate the antioxidant effect of Nigella sativa extract on motility, plasma membrane function, mitochondrial membrane potential (MMP), DNA damage, and intracellular ROS production. Methods: A total of 20 sperm samples were used. Samples were divided into six experimental groups, including groups with aqueous extract from N. sativa seeds at concentrations of 1% to 6%, a cryopreserved control group, and a fresh control group. Results: Statistical analysis showed significantly higher total sperm motility at concentrations of 3% to 6% than in the vitrified semen control group. Additionally, progressive motility and all motion characteristics at all concentrations were significantly higher than in the vitrified semen control group. The presence of N. sativa seed extract also improved the quality of the sperm parameters assayed in all experimental groups (1%-6%; intracellular ROS production, DNA damage, MMP, and sperm membrane function) compared to the control group. Conclusion: Higher concentrations of N. sativa led to improvements in all sperm parameters and sperm quality. These findings indicate that N. sativa seed extract is effective for improving the quality of sperm after vitrification.