Earticle

Fipronil is widely used pesticide for insects, such as granular turf products, pet care flea and tick solutions. Although the Fipronil presents moderately risky, only for several researches have been reported that the Fipronil affects fertility of insects, fish, and so on. However, effects of Fipronil on mammalian fertility are not fully understood yet. Therefore, the aims of this study are investigating the effects of Fipronil on sperm functions. Spermatozoa were incubated with various concentrations of Fipronil (0, 0.1, 1, 10, 100, and 300 μM). Then sperm motility and motion kinematics were evaluated using computer-assisted sperm analysis. In addition, capacitation status and intracellular ATP level were evaluated using combined Hoechst 33258/chlortetracycline fluorescence and ATP Assay kit, respectively. Sperm motility, rapid speed, progressive, curvilinear velocity, average path velocity, and mean amplitude of head lateral displacement were significantly decreased in higher concentration of Fipronil (1, 10, 100, and 300 μM) treatments. Beat-cross frequency was significantly decreased in presence of 10, 100, and 300 μM Fipronil. Straight-line velocity and the acrosome reaction were significantly decreased and increased in 100 and 300 μM Fipronil treatments, respectively. In addition, the highest concentration of Fipronil (300 μM) was significantly decreased capacitation status and intracellular ATP level. Our results showed that Fipronil suppress sperm motility, motion kinematics, the acrosome reaction, capacitation status and intracellular ATP level. Therefore, Fipronil can negatively affect male fertility and the concentration of Fipronil need to be controlled when Fipronil be used for pesticide.

Peroxiredoxins (PRDXs), which are essential antioxidant enzymes for physiological activity, have reported that influence sperm function and consequently male fertility. Although PRDXs are well known as important factor to affect male fertility, there has been a lack of attempt to discover the accurate role and mechanism of PRDXs in male fertility. Therefore, the principal objective of this study is to elucidate the role of PRDXs in sperm function and fertilization. We treated mouse spermatozoa with different dose of specific inhibitor of PRDXs, conoidin A (1, 10, or 100 μM). Our results revealed that conoidin A significantly decreased oxidized form of PRDXs (PRDXs-SO3) in mouse spermatozoa. Inhibited PRDXs activity are then resulted in a significant decrease in sperm motility/motion kinematics, viability, mitochondrial membrane potential, and intracellular ATP levels. On the other hand, intracellular levels of ROS and DNA fragmentation index were significantly increased following exposure to conodin A. Next, we evaluated capacitation and acrosome reaction status, and subsequently tyrosine phosphorylation and protein kinase-A activity to investigated underlying mechanism of PRDXs on sperm capacitation status. Capacitation and acrosome reaction were significantly decreased perhaps due to reduction of tyrosine phosphorylation and protein kinase- A activity. Finally, we investigated the effect of PRDXs on fertilization and early embryonic development. Our results described that decreased PRDXs activity significantly decreased fertilization and early embryonic development. Consequently, we demonstrated that inhibition of PRDXs activity has a direct impact on male fertility via decreased important physiological sperm function, eventually resulted in poor fertilisation and embryonic development.

The current study was designed to evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase system (XO) on sperm function and DNA fragmentation in porcine spermatozoa. ROS were produced by a combination of 1,000 μM X and 50 mU/ml XO. The ROS scavengers such as superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without antioxidants at 37℃ under 5% CO2 incubator. Ca-ionophore-induced acrosome reaction, the proportion of swollen spermatozoa under hypo-osmotic condition, malondialdehyde formation for the analysis of lipid peroxidation, and the proportion of DNA fragmentation were determined after 2 hours incubation. The action of ROS on porcine spermatozoa resulted in decreased Ca-ionophore-induced acrosome reaction and membrane integrity, increased the formation of malondialdehyde, and the proportion of sperm with DNA fragmentation(p<0.05). The toxic effects caused by ROS were completely alleviated by CAT in terms of sperm function and characteristics, however SOD did not serve the same scavenger effect as CAT. To conclude, the ROS can cause significant damage to porcine sperm functions and characteristics, which can be minimized by the use of antioxidants.

Temephos is recommended a non-systemic organophosphorus insecticide by WHO because it is safe for humans and has low toxicity to mammals compared with other chemical compounds such as chlorine, potassium permanganate, zinc carbamate, and dichlorodiphenyltrichloroethane (DDT). However, Temephos is involved in the hazardous pesticide category PAN Bad Actor (Pesticide Action Network, USA). Moreover, no study was performed to evaluate toxicity of Temephos in mammalian germ cell. The objectives of this study was to assess the effect of Temephos on sperm functions. First, caudal epididymis spermatozoa from ICR mice were incubated with Temephos (0, 0.1, 1, 10, and 100 μM). Then sperm motility and motion kinematics were assessed using computer-assisted sperm analysis. In addition, capacitation status and adenosine triphosphate (ATP) level were assessed using combined Hoechst 33258/chlortetracycline fluorescence and ATP detection kit, respectively. The acrosome reaction and capacitation status were significantly increased and decreased in the highest concentration of Temephos (100 μM), respectively. In addition, ATP levels in spermatozoa were significantly decreased in higher concentration of Temephos (1, 10, and 100 μM). However, Sperm motility and motion kinematics (motility, medium, slow, progressive, hyperactivated, curvilinear velocity, straight-line velocity, average path velocity, beatcross frequency, dance mean, amplitude of lateral head displacement) were not significantly altered with Temephos. The results were shown that Temephos affect capacitation status, the acrosome reaction and ATP level. Taken together, we suggest that the present study may be applied to evaluate risk of Temephos on male fertility.

The aim of this study was to evaluate effect of Propolis on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryo-preserved in 11% lactose with 20% egg yolk (LEY) and Triladyl containing propolis (0, 10, 30, and 50 ng/mL) with 0.05% ethanol. Cryo-preserved boar sperms was thawed at 37.5℃ for 45 sec in water-bath. Sperm viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed sperm, which was cryo-preserved using LEY and triladyl, was decreased in 0, 30, and 50 propolis treatment groups. However, there was no significant difference. Acrosome reaction and mitochondrial intact of sperm was not influenced by all of propolis treatment using both of freezing extender. Motility of frozen-thawed sperm with LEY containing 10 ng/mL propolis was significantly higher than in 0, 10, and 50 ng/mL propolis treatment groups (p<0.05), whereas there was no significant difference compared to control. Especially, 0 and 30 ng/mL propolis treatment groups were significantly decreased compared to control (p<0.05). Similar to result in frozen-thawed sperm with LEY, motility of frozen-thawed sperm with triladyl was significantly increased in 10 ng/mL propolis treatment groups compare to control and other propolis treatment groups (p<0.05). In conclusion, 10 ng/mL propolis improves mobility in frozen-thawed boar sperm with LEY and triladyl, whereas 30 and 50 ng/mL propolis reduced motility. But, the addition of propolis during cryopreservation did not influence to the membrane function of frozen-thawed spermatozoa. Therefore, it is necessary to continue the study on the effects of propolis on boar spermatozoa during cryopreservation.

Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin and may participate in mammalian fertilization. Although correlations have been reported between reactive oxygen species (ROS) and sperm function, the relationship between PA activity and ROS is unknown. We determined the effects of ROS on sperm function and PA activities in boar spermatozoa preincubated under the X-XO system. When spermatozoa were treated with the X+XO group, a significant increase (p<0.05) was observed in the percentage of acrosome reacted spermatozoa compared with that of the control group. However, when antioxidants were added to the medium with X+XO, the rate of acrosome reaction tended to decrease. Also, a significantly lower percentage of acrosome reacted spermatozoa was observed in the X+XO+catalase group at 6 hr of incubation compared with that of X+XO group. The density of malondialdehyde (MDA) was higher in the X+XO group than in different treatment groups. In another experiment, incubation of spermatozoa in medium with X+XO was associated with a significant (p<0.05) increase in activity of tPA-PAI and tPA compared with the control group. Antioxidants decreased the increased activity of tPA-PAI and tPA by preincubation in the X-XO system. Also, a significantly lower (p<0.05) activities of tPA-PAI and tPA were observed in the X+XO+catalase group compared with the X+XO group. No significant differences, however, were observed in the activity of uPA. These results suggest that the increase of acrosome reaction by the X-XO system resulted in increase of PAs activity in the sperm incubation medium.

본 연구는 인공수정 센터에서 이용되고 있는 종모돈의 인공수정 분만율의 차이를 기준으로 하여 정액의 보존 일에 따른 정자의 생존율, CASA, HOST, IVP 및 SCSA를 조사하여 이들 정자의 분석법들과 인공수정 분만율과의 상관관계를 조사하여 종모돈의 수정 능력을 예측할 수 있는 정액 평가법을 개발하고자 실시하였다. 종모돈의 액상정액을 대상으로 CASA, HOST, IVP 및 SCSA 기법을 적용한 정액의 평가 결과는 다음과 같았다. 정액의 보존 일별 HOST 결과는 보존 일이 경과함에 따라 유의적으로 감소하였고(P<0.05), IVP 결과 난자 당 침투 정자 수는 보존 3일째에 유의적으로 감소하였다(P<0.05). 보존 0일과 6일에 HOST는 분만율이 80% 이상인 종모돈 군이 70% 미만인 종모돈 군 보다 유의적으로 높았고, 3일에서는 80% 이상인 종모돈 군이 다른 종모돈 군에 비하여 높았다(P<0.05). IVP는 보존 0일에 분만율이 80% 이상인 종모돈 군이 70% 미만인 종모돈 군보다 난자당 침투 정자 수가 유의적으로 많았다(P<0.05). %Red는 보존 0일과 3일에는 분만율이 80% 이상과 70% 미만인 종모돈 군 간에 유의적인 차이를 보였으나 보존 6일에서는 모든 종모돈 군간에 유의적인 차이가 있었다(P<0.05). %Red(r=0.79, P<0.01 ; r=0.86, P<0.01 ; r=0.88, P<0.01)는 정액의 보존 일이 증가함에 따라 분만율과의 부의 상관 계수가 증가하였다. 이상의 결과를 종합해 보면 SCSA는 정액의 수정 능력을 평가하는데 있어서 매우 유용한 방법이며, 정액의 보존 일은 여러 가지 정자 기능과 SCSA 결과에 크게 영향을 주는 것으로 나타났다. 정액의 수정 능력을 평가함에 있어서 정액의 보존 일령을 고려하고 정자 기능 검사와 SCSA를 병행하여 실시할 경우 종모돈의 수정 능력 평가 시 정확성을 더욱 높일 수 있는 유용한 방법이 될 수 있을 것으로 사료된다.

author : C. K. Kim, Dep. of Animal Science and Technology, College of Industial Sciences, Chung-Ang Universit Ansung-Si Kyunggi-Do 456-756 Korea.Tel : 031-670-3026, E-mail : ckkim7503@cau.ac.kr

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol (39.85% ±11.41) than in 5% glycerol treatment (18.08%±1.61). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol (34.12%±11.02) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

남성 난임은 남성 생식 생리와 전반적인 생식 건강을 방해하는 내분비계 교란 물질에 대한 노출 증가로 인해 증가하고 있다. Butylparaben(BP)은 p-hydroxybenzoic acid의 alkyl ester로, 강한 항균 활성을 나타내며 식품, 화장품 및 개인 위생용품에서 방부제로 널리 사용되고 있다. 이전 연구들에서 BP가 인체 건강에 부정적인 영향 을 미칠 수 있다는 가능성이 제기되었으나, 남성 생식 독 성에서의 역할에 대한 근거는 아직 부족한 실정이다. 따 라서 본 연구에서는 BP의 남성 생식 독성을 평가하기 위 해 돼지 정액을 0, 100, 200, 300μM BP로 처리한 후 정 자 운동성, 생존율 및 미토콘드리아 기능을 분석하였다. 정자 운동성은 컴퓨터 지원 정자 분석 시스템을 사용하 여 분석되었으며, BP 노출은 운동성(89.13±2.98 vs. 39.47±7.05)과 여러 운동 역학적 매개변수를 변화시켰다. SYBR-14/PI 이중 염색으로 평가한 정자 생존율은 BP 처 리에 의해 감소하였다(79.09±2.71 vs. 68.09±2.78). 또한 BP 노출은 미토콘드리아 막 전위와 MitoTracker 염색을 감소시켰으며(각각 74.24±2.47 vs. 61.11±3.21, 1.00±0.04 vs. 0.73±0.04), 반면 MitoSOX 수준은 증가하였다 (1.00±0.00 vs. 1.13±0.02). 이는 미토콘드리아 산화 스트 레스 증가와 기능 이상을 시사한다. 종합적으로, 이러한 결과는 BP가 정자 운동성 저하, 생존율 감소, 그리고 미 토콘드리아 기능 장애를 통해 남성 생식 독성을 유도함 을 시사한다.

Male infertility has been increasing due to greater exposure to endocrine disrupting chemicals, which interfere with male reproductive physiology and overall reproductive health. Butylparaben (BP), an alkyl ester of p-hydroxybenzoic acid, exhibits strong antimicrobial activity and is widely used as a preservative in foods, cosmetics, and personal care products. Despite the findings of previous studies indicating a potential link between BP and adverse effects on human health, evidence regarding its role in male reproductive toxicity is still scarce. Therefore, to evaluate the male reproductive toxicity of BP, porcine semen was treated with 0, 100, 200, and 300 μM BP, followed by assessment of sperm motility, viability, and mitochondrial function. Sperm motility was analyzed using a computer-assisted sperm analysis system, and BP exposure altered motility (89.13±2.98 vs. 39.47±7.05) as well as multiple kinematic parameters. Sperm viability, assessed using SYBR-14/PI dual staining, was reduced by BP treatment (79.09±2.71 vs. 68.09±2.78). BP exposure also decreased mitochondrial membrane potential and MitoTracker labeling (74.24±2.47 vs. 61.11±3.21 and 1.00±0.04 vs. 0.73±0.04, respectively), while increasing MitoSOX levels (1.00±0.00 vs. 1.13±0.02), indicating elevated mitochondrial oxidative stress and dysfunction. Collectively, these results suggest that BP induces male reproductive toxicity by impairing sperm motility, reducing viability, and disrupting mitochondrial function.

본 연구는 돼지 정자 동결시 taurine과 ａ-tocopherol의 첨가가 융해후 정자 성상과 정자 기능 활성산소계(reactive oxygen species; ROS)의 발생 정도 및 지질 산화(lipid peroxidation, LPO)에 미치는 영향을 구명하기 위하여 시행하였다. 1차 및 2차 희석액내 taurine과 ａ-tocopherol이 첨가된 돼지 동결정액의 융해 후 정자 운동성 양상, 정자 생존성, 정자 기법을 적용하여 다음과 같은 결과를 얻었다. Taurine(25mM, 50mM), ａ-tocopherol(500㎛, 1,000㎛) 단일처리군 그리고 taurine과 ａ-tocopherol의 혼합처리군(25mM~500㎛, 50mM~1,000㎛)은 동결ㆍ융해 후 대조군과 비해 정자 운동성과 생존성은 유의적인 차이를 나타내지 않았다. Taurine과 ａ-tocopherol의 혼합처리군 50mM~1,000㎛에서 HOST, 점체 반응이 대조군과 비교하여 유의차는 인정되지 않았으나 다소 증가하였다. 동결ㆍ융해 정자의 ROS 발생 억제를 위한 taurine과 ａ-tocopherol의 모든 처리군은 대조군과 비교하여ㆍO₂- 발생을 유의적으로 완화시키지 못하였다. 그러나 taurine 단일처리군(25mM), ａ-tocopherol 단일처리군(50mM,1,000㎛)과 혼합처리군(25mM~50mM,50mM~1,000㎛)은 H₂O₂의 발생을 대조군과 비교하여 유의적으로 완화시켰다(P<0.05). 동결ㆍ융해 정자의 malondialdehyde의 생산은 taurine과 ａ-tocopherol의 모든 처리군에서 대조군과 비교하여 유의적인 감소를 보였다(P<0.05). 이상의 결과를 종합해 보면 돼지 정액의 동결보존시 taurine와 ａ-tocopherol 같은 항산화제의 처리는 ROS 발생과 LPO을 효과적으로 완화시킴에 따라 돼지 정액의 동결보존 효율을 증진시킬 수 있는 유용한 방법이라 사료된다.

The present study evaluated whether an exogenous antioxidants, taurine and ａ-tocopherol, could, when added to the freezing extender, improve the post-thaw sperm characteristics, function, the level of reactive oxygen species (ROS) generation, and the level of lipid peroxidation (LPO) in frozen-thawed porcine semen. CASA (computer-aided sperm analysis), HOST (hypoos-motic swelling test), chemiluminescence using luminol and lucigenin and the detection of malondialdehyde for LPO was performed in frozen-thawed porcine sper-matozoa. The results obtained in these studies are as follows. While no beneficial effects of taurine and ａ-tocopherol supplementation were visible in motility, viability, acrosome reaction, tail swelling patterns, and the generation of O₂- of frozen-thawed porcine sper-matozoa, H₂O₂ was decreased by all treatments except taurine 50mM treatment. In conclusion the taurine and ａ-tocopherol treatments during freezing reduced generation of reactive oxygen species and production of malondialdehyde in frozen-thawed porcine semen, and the ROS savangers may minimize various damages of spermatozoa during freezing.

Background: Estrogen signaling pathways are modulated by exogenous factors. Panax ginseng exerts multiple activities in biological systems and is classified as an adaptogen. Zearalenol is a potent mycoestrogen that may be present in herbs and crops arising from contamination or endophytic association. The goal of this study was to investigate the impact of P. ginseng, zearalenol and estradiol in tests on spermatozoal function. Methods: The affinity of these compounds for estrogen receptor (ER)-alpha and beta ($ER{\alpha}$ and $ER{\beta}$)-was assessed in receptor binding assays. Functional tests on boar spermatozoa motility, movement and kinematic parameters were conducted using a computer-assisted sperm analyzer. Tests for capacitation, acrosome reaction (AR), and chromatin decondensation in spermatozoa were performed using microscopic analysis. Results: Zearalenol-but not estradiol ($E_2$)- or ginseng-treated spermatozoa-decreased the percentage of overall, progressive, and rapid motile cells. Zearalenol also decreased spontaneous AR and increased chromatin decondensation. Ginseng decreased chromatin decondensation in response to calcium ionophore and decreased AR in response to progesterone ($P_4$) and ionophore. Conclusion: Zearalenol has adverse effects on sperm motility and function by targeting multiple signaling cascades, including $P_4$, $E_2$, and calcium pathways. Ginseng protects against chromatin damage and thus may be beneficial to reproductive fitness.

정액 동결 방법들은 지속적으로 향상되어 왔지만 융해 속도에 대한 연구는 여전히 정립되어 있지 않다. 따라서, 본 연구의 목적은 동결 후 융해 속도가 정액의 기능에 미치는 영향을 알아보고자 하였다. 비글견으로부터 채취된 정액은 동결 보존 후 다른 융해 속도 ($37^{\circ}C$/1분 or $70^{\circ}C$/15초)에서 융해 되었다. 융해 후, 운동성, 생존성, 정상 형태율, 형질막 온전성, phosphatidylserine (PS) translocation, 세포내 $H_2O_2$ 수준을 평가하였다. $70^{\circ}C$에서 융해된 정자는 $37^{\circ}C$에서 융해된 정자에 비해 향상된 정자 운동성, 생존성, 정상 형태율, 세포막 온전성, non-PS translocation을 보였으나(P < 0.05), $70^{\circ}C$와 $37^{\circ}C$에서 융해된 실험군간 세포내 $H_2O_2$ 수준에서 유의적인 차이는 나타나지 않았다(P > 0.05). 결론으로, $70^{\circ}C$에서의 융해는 개 정자 동결 후 정자 기능을 증진시켰으며, 적절한 융해 온도는 동결 정자의 기능을 향상시킬 수 있을 것으로 판단된다.

Sperm cryopreservation methods have been improved over the last few decades. However, an optimized thawing rate has not yet been established. Therefore, we investigated the effect of thawing rate on sperm function after cryopreservation. The ejaculates collected from beagle dogs were cryopreserved and then thawed at two different thawing rates ($37^{\circ}C$ for 1 min or $70^{\circ}C$ for 15 sec). The thawed sperm were evaluated for motility, viability, morphology, plasma membrane integrity, phosphatidylserine (PS) translocation, and intracellular $H_2O_2$ level. The sperm thawed rapidly at $70^{\circ}C$ showed improved motility, viability, normal morphology, plasma-membrane integrity and non-PS translocation compared to the sperm thawed slowly at $37^{\circ}C$ (P < 0.05). However, the intracellular $H_2O_2$ levels were not significantly different between the rapid- and slow-thawed sperm (P > 0.05). In conclusion, sperm rapid thawing at $70^{\circ}C$ could improve the function of cryopreserved canine sperm, and the appropriate thawing rate would enhance the quality of the cryopreserved sperm.

Objective : To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. Materials and Methods: ROS were produced using a combination of 1000 uM X and 50 mU/ml XO. The ROS scavengers: superoxide dismu tase (SOD) (200 U/ml) and catalase (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at $37^{circ}C$ under 5% $CO_2$ incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Caionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. Results: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. Conclusion: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.