• Asian-Australasian Journal of Animal Sciences
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• 제13권1호
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• pp.10-18
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• 2000

This paper describes the magnetic orientation of the intact and demembranated bull sperm treated by DTT or heparin in a 5,400 G 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 sperm head area at 1, 3, and 6 days. After measuring the area, each sperm sample was exposed to a 5,400 G static magnetic field generated by Nd-Fe-B permanent magnets for 24 hours at room temperature. Results showed that the decondensation of bull sperm nuclei was not induced by the heparin treatment, however, incomplete decondensation was induced by the DTT treatment. During the magnetic orientation, bull sperms treated by DTT or heparin had low percentages of long axis perpendicular to the magnetic lines of force. However, different aspects were obtained for long axis perpendicular orientations following treatment of DTT or heparin. Through the DTT treatment, the decline of long axis perpendicularly oriented percentages was due to the increase of long axis parallel orientation with the head of the flat plane perpendicular to the magnetic lines of force, whereas, using the heparin treatment, the decline of long axis perpendicular orientation was due to the increment of long axis parallel orientation with the head of the flat plane parallel to the magnetic lines of force. Also, percentages of the head of the flat plane perpendicular were decreased by the heparin treatment. These findings suggest that maintaining the structure of protamine in the chromatin is necessary for the sperm head to orient with its flat plane perpendicular, and maintaining the disulfide bond in the chromatin is necessary for the long axis of sperm to orient perpendicularly.

• 생명과학회지
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• 제33권7호
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• pp.586-592
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• 2023

돼지 정액을 저장하는 동안 산화스트레스의 발생은 정자의 질과 생존에 영향을 미치는 중요한 인자이다. 정액의 저장은 온도 변화, 동결보호제 등의 다양한 스트레스 인자에 노출되어 있다. 이러한 정자 내에서의 산화스트레스는 활성산소종의 생성에 의해 발생되며, 이는 지질, 단백질, DNA와 같은 세포를 구성하는 물질에 산화적으로 손상을 일으킨다. 활성산소종과 항산화물질의 균형있는 체계는 정자의 생존과 그 기능을 유지하는 데 중요한 역할을 한다. 정액을 장기간 보존하게 되면 활성산소종의 수준이 증가하여 정자의 운동성, 막 온전성, DNA 온전성에 영향을 미치게 된다. 또한, 활성산소종에 의해 유도된 지질과산화 반응은 정자막의 유동성과 안정성에 영향을 미쳐 정자의 운동성을 감소시킨다. 그리고, DNA의 산화적 손상은 DNA 단편화를 일으켜 정자의 DNA 온전성을 손상시킬 수 있다. 결론적으로, 정액을 보관하는 동안 발생되는 산화스트레스는 정자의 질과 기능을 유지하는 데 중요하다. 따라서, 산화스트레스의 기본적인 메커니즘과 정자의 기능에 미치는 영향을 이해하는 것은 산화스트레스로부터의 손상을 최소화하고, 효율적이고 기능적인 정자의 저장 방법을 개선하기 위한 효과적인 전략과 연구 개발을 위해 중요할 것으로 판단된다.

• Clinical and Experimental Reproductive Medicine
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• 제36권1호
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• pp.45-53
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• 2009

목 적: 본 연구는 정상 정액을 배양액으로 세척한 후 $4^{\circ}C$, $22^{\circ}C$, $37^{\circ}C$에서 5일 동안 보존하면서 정자의 운동성, 생존성을 관찰하여 정자의 운동성과 수명 유지를 위한 적정 배양 환경을 분석하고자 하였다. 연구방법: 정액검사 시 정상으로 판정된 남성의 정자를 HTALP 배양액으로 세척하여 정자의 최종 농도 $1{\times}10^6/ml$로 각 5 ml을 배양온도 $4^{\circ}C$, $22^{\circ}C$, $37^{\circ}C$에서 5일 동안 배양하였다. 1일, 3일 5일째에 CASA에 의해 운동성을 측정하였고, HOST로 정자막의 온전성을 분석하였으며, CTC pattern으로 수정능획득 상태 분석하여 최적의 배양 환경을 분석하였다. 결 과: 정자의 운동성, 생존성 및 수정능획득이 야기되지 않은 정자는 배양일에 따라 유의하게 감소하였다 (p<0.05). 또한 정자 배양 후 1일에는 정자의 운동성, 생존성 및 정자막의 온전성과 CTC pattern은 온도에 따라 차이가 없었으나, 배양 후 3일과 5일에서는 $22^{\circ}C$에서 배양된 정자가 다른 배양온도에 비해 가장 잘 보존되었다 (p<0.05). 결 론: HTALP로 세척된 정자를 $22^{\circ}C$에서 보존 시 5일까지 정자의 운동성과 수명을 보조생식술에 적합한 수준으로 유지시킬 수 있는 최적 배양 환경으로 제시할 수 있다.

• Animal Bioscience
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• 제37권6호
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• pp.1001-1006
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• 2024

Objective: This study aimed to investigate the effect of Codonopsis pilosula polysaccharide (CPP) on the motility, mitochondrial integrity, acrosome integrity rate, and antioxidant ability of sheep sperm after preservation at 4℃. Methods: Semen from healthy adult rams were collected and divided into four groups with separate addition of 0, 200, 400, and 1,000 mg/L CPP. Sperm motility was analyzed using the Computer-Assisted Semen Analysis software after preservation at 4℃ for 24, 72, 120, and 168 h. Sperm acrosome integrity rate was analyzed by Giemsa staining at 24, 72, and 120 h, and mitochondrial membrane integrity was analyzed by Mito-Tracker Red CMXRos. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of spermatozoa were measured after 120 h of preservation. Results: The sperm viability and forward-moving sperm under 200 mg/L CPP were significantly higher than that in the control group at 72 h (61.28%±3.89% vs 52.83%±0.70%, 51.53%±4.06% vs 42.84%±1.14%), and 168 h (47.21%±0.85% vs 41.43%±0.37%, 38.68%±0.87% vs 31.68%±0.89%). The percentage of fast-moving sperm (15.03%±1.10% vs 11.39%±1.03%) and slow-moving sperm (23.63%±0.76% vs 20.29%±1.11%) in the 200 mg/L group was significantly higher than control group at 168 h. The mitochondrial membrane integrity of the sperm in the group with 200 mg/L CPP was significantly higher than those in the control group after storage at 4℃ for 120 h (74.76%±2.54% vs 65.67%±4.51%, p<0.05). The acrosome integrity rate in the group with 200 mg/L (87.66%±1.26%) and 400 mg/L (84.00%±2.95%) was significantly higher than those in the control group (80.65%±0.16%) after storage for 24 h (p<0.05). CPP also increased T-AOC and decreased the MDA concentration after preservation at 4℃ (p<0.05). Conclusion: Adding CPP could improve the T-AOC of sperm, inhibit lipid peroxidation, and facilitate semen preservation.

• 생명과학회지
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• 제27권5호
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• pp.517-523
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• 2017

본 실험은 항산화제인 melatonin, silymarin, curcumin 및 vitamin E가 aresenite에 의해 손상된 돼지의 정자성상능력에 미치는 영향을 조사한 연구이다. 돼지정자는 채취 후 희석하여 실험에 이용하였으며, $100{\mu}M$ arsenite는 인위적으로 정자를 손상시키는데 사용하였다. 또한 100 nM melatonin, $2{\mu}M$ silymarin, $10{\mu}M$ curcumin 및 $500{\mu}M$ Vitamin E를 희석된 돼지정자에 첨가하여 3, 6 및 9시간 동안 배양 후 정자의 운동성, 원형질막 온전성, 미토콘드리아 기능성 및 원형질막 지질과산화를 검토하였다. 그 결과, 정자의 운동성 및 원형질막 온전성은 $100{\mu}M$ arsenite 처리구에서 유의적으로 감소하였으며, 항산화제 단독처리구는 유의적으로 증가하였다. 또한, arsenite에 의해 손상된 처리구는 항산화제에 의해 유의적으로 증가하였다(p<0.05). 정자의 미토콘드리아 기능성은 $100{\mu}M$ arsenite 처리구에서 유의적으로 감소하였고, 정자의 원형질막 지질과산화는 $100{\mu}M$ arsenite 처리구에서 유의적으로 증가하는 것으로 나타났다(p<0.05). 결론적으로, arsenite의해 감소된 돼지정자의 운동성과 원형질막은 melatonin, silymarin, curcumin 및 vitamin E와 같은 항산화제에 의하여 예방될 수 있다고 판단되며, arsenite에 의해 감소된 정자의 수정능력은 항산화제에 의해 회복될 수 있을 것이라 생각된다.

• 한국수정란이식학회지
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• 제26권1호
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• pp.71-78
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• 2011

This study was undertaken to find out the effect of cholesterol and serum albumin on sperm ability and lipid peroxidation levels period to the liquid storage of miniature pig sperm. Ejaculated semen from miniature pigs was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle, and extended with Modena solution {with and without BSA, methyl-beta-cyclodextrin (-cholesterol) and cholesterol loaded cyclodextrin (+cholesterol)}. Each semen was assessed for viability (SYBR-14/PI staining) and acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining at 1, 3, 5, 7 and 10 days of storage. At for the effects of cholesterol and serum albumin on lipid peroxidation, semen were incubated with $H_2O_2$ ($10\;{\mu}M$), and lipid peroxidation level were measured by flow cytometry using the lipid peroxidation reporter probe $C_{11}-BODIPY^{581/591}$. The result, lipid peroxidation level in sperm added with cholesterol were lower in $10\;{\mu}M$ $H_2O_2$ compared to the added sperm with serum albumin. Also, added cholesterol to sperm had significant (p<0.05) higher viability when storage for 7 and 10 days and lower when 10 days of storage percentage of acrosome-reacted sperm (AR pattern) in acrosome state as say result compared to other treated groups. In conclusion, role of cholesterol during lipid storage in miniature pig spermatozoa was protected boar spermatozoa from lipid peroxidation prior to lipid storage. Addition serum albumin during lipid storage in sperm may be induce sperm membrane damage by lipid peroxidation. Therefore, addition of cholesterol to miniature pig sperm will be lead to extension of liquid storage periods.

• 한국양식학회지
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• 제17권1호
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• pp.46-50
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• 2004

In the present study, attempts were made to cryopreserve sea urchin, Anthocidaris crassispina sperm in liquid nitrogen, to evaluate the effects of various cryoprotectants and freezing rates on motility, survival rate and fertilization rate of the post-thawing sperm, and the ultrastructural changes of sperm after cryopreservation were observed. The highest values of sperm motility (motility index: 3.3$\pm$0.37) and survival rate (72$\pm$3.5%) were obtained with 15% dimethyl sulfoxide (DMSO), and these values were significantly higher than those of sperm preserved with glycerol. Comparisons of motilities and survival rates between treatments of difference freezing rates showed that there was no difference between procedures (a) 5$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.3$\pm$0.31 ; survival late 70$\pm$2.7%) and (b) 3$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.1$\pm$0.29; survival rate 69$\pm$3.7%), while the results of (c) 1$0^{\circ}C$/min to -8$0^{\circ}C$ were significantly lower than the others (motility index: 2.2$\pm$0.33 ; survival rate 42$\pm$4.6%). There was no significant difference in fertilization rate between fresh sperm and sperm preserved with 15% DMSO as cryoprotectant and freezing rate (3$0^{\circ}C$/min to -8$0^{\circ}C$). Some ultrastructural changes of sperm, such as the detachment of plasma membrane, the destruction of mitochondria, and the flagellum rolling up head, were observed after cryopreservation. Morphological normality of the sperm in 15% DMSO frozen at the ratio of 5$0^{\circ}C$/min to -8$0^{\circ}C$ was better than the others.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.181-189
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• 2011

Cryopreservation protocols induce partially irreversible damage to mammalian sperm plasma membranes. Previous studies have indicated that adding cholesterol to the plasma membrane, as cholesterol-loaded-cyclodextrins, improves cryosurvival of sperm. Therefore, the purpose of this study was to determine if treating sperm of Markhoz bucks with cholesterol-loaded-cyclodextrins (CLC) (0, 0.75, 1.5, 2.25 and 3 mg/ml diluted $240{\times}10^6$ sperm/ml) in Tris-citric acid-glucose diluents with and without egg yolk (containing 5% glycerol) would improve the post-thaw sperm quality. The motion characteristics were evaluated with a Computer Assisted System Analyzer (CASA); acrosome integrity and vitality were measured with the triple-stain technique. Samples were recovered before and after freezing by means of putting straws into $37^{\circ}C$ water for 30 sec and then parameters were assessed. The results showed that the treatments significantly affected motility, progressive motility, recovery rate, curvilinear velocity, beat cross frequency, live sperm with reacted acrosome, live sperm with unreacted acrosome, dead sperm with reacted acrosorne, and dead sperm with unreacted acrosome during freezing (p<0.05). However; no significant differences were found for average path velocity, straight line velocity, amplitude of lateral head displacement, straightness and linearity (p>0.05). The best results were observed for extender containing 2.25 mg/ml ($240{\times}10^6$ sperm/ml) CLC supplemented with 2.6% egg yolk. In conclusion, the findings of this study indicate improved Markhoz sperm viability and motility following treatment in the presence of egg yolk.

• Clinical and Experimental Reproductive Medicine
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• 제46권2호
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• 한국임상수의학회지
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• 제34권2호
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.