• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

• Journal of Animal Science and Technology
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• v.54 no.4
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• pp.283-290
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. The objective of this study was to investigate the effect of taurine, hypotaurine and trehalose as antioxidants on the function of the freezing-thawed sperm in Korean Jeju Black Bull. The semen was cryopreserved with tris egg yolk extendercontaining 7% glycerol and treated with 20mM taurine, hypotaurine and trehalose. Frozen-thawed sperms were evaluated for sperm motility, viability, membrane integrity, acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender containing 7% glycerol only as control. Frozen-thawed semen evaluation clearlyindicated that the addition of taurine or hypotaurine significantly improved (p<0.05) the motility and viability compared to control spermatozoa. Moreover, in membrane integrity, swollen sperm ratio was significantly increased (p<0.05) in taurine, hypotaurine or trehalose compared to control. In sperm acrosome integrity, F pattern ratio was increased (p<0.05) in hypotaurine among treatments, and AR pattern was significantly lowered (p<0.05) in taurine, hypotaurine and trehalose. In assessed sperm fertilizing ability, taurine, hypotaurine or trehalose significantly improved (p<0.05) the ratio of pronucleus formation and SFI. Finally, compared with the control, addition of taurine, hypotaurine or trehalose as an antioxidant to the freezing extender showed more positive effects on the frozen-thawed spermatozoa. It is concluded that the addition of taurine, hypotaurine, or trehalose to the freezing extender could reduce cryodamage of the Korean Jeju Black Bull spermatozoa.

• Journal of Animal Science and Technology
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• v.49 no.6
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• pp.729-736
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• 2007

The objective of this study was to investigate the anti-oxidative effects of pyruvate, taurine and melatonin on sperm characteristics(motility, membrane integrity) and lipid peroxidation(LPO) for in vitro storage of boar semen. Semen was treated with various antioxidants such as pyruvate(1mM), taurine(50mM) and melatonin(100nM) with or without 100uM H2O2. Antioxidant treatments were significantly increased the sperm motility when compare to control group in all incubation periods(P≤0.05). Hypoosmotic swelling test(HOST), membrane integrity was similar to the result of motility. In lipid peroxidation measurement by TBA reactions of spermatozoal plasma membrane, malondialdehyde(MDA) level in control and antioxidant treatments were lower than those of antioxidant plus H2O2 or H2O2 treatment for 3 to 6 h incubation period. Relationships of evaluation methods for sperm viability were investigated by motility, membrane integrity and lipid peroxidation. Among evaluation methods, LPO vs motility and membrane integrity vs LPO were negatively correlated(－0.23~－0.92 and －0.68~－0.85), but membrane integrity vs motility was positively correlated (0.53~0.94) in all treatments. These experiments indicate that supplementation of antioxidant to the semen extender can increase the sperm motility and membrane integrity and decrease the lipid peroxidation of spermatozoal plasma membrane. The HOST might be utilized to evaluate the sperm quality instead of lipid peroxidation or motility.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.1
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• pp.14-21
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• 2019

Objective: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. Methods: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). Results: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r = 0.70 and r = 0.68, respectively; p< 0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r = 0.77, p< 0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r = 0.59, r = 0.57, and r = 0.72, respectively; p< 0.001). Conclusion: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.99-105
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• 2009

Curcumin is a major active component of the food flovour tumeric. It has been used for the treatment of many diseases such as inflammatory and infectious diseases, cancer and other disease due to its antioxidant properties. Curcumin is a powerful scavenger of many free radicals such as superoxide anion, hydroxyl radical and nitric oxide. The objective of this study was to investigate the antioxidative effects of curcumin against hydrogen peroxide on semen quality during in vitro storage of boar semen. The sperm treated with different concentration of curcumin (1, 5 and 10 ${\mu}M$) in the presence or absence of hydrogen peroxide (250 ${\mu}M\;H_2O_2$) were incubated for 3, 6 and 9 hr at $37^{\circ}C$ and analyzed sperm characteristics such as motility, membrane integrity (MI), lipid peroxidation (LPO), reactive oxygen species (ROS) and DNA fragmentation (DF). The sperm motility and MI in $H_2O_2$ treated group ($47.8%{\pm}6.8$ and $24.8%{\pm}2.2$) were significantly decreased when compare to curcumin treated group ($79.8%{\pm}2.7$ and $34.6%{\pm}1.0$, respectively) irrespective of incubation periods(p<0.05). The LPO of spermatozoal plasma membrane was measured by thiobarbituric acid (TBA) reactions for malondialdehyde (MDA), MDA level in control ($11.6{\pm}0.6\;nmol/L{\times}10^6$) and curcumin groups ($10.7{\pm}0.3\;nmol/L{\times}10^6$) were lower than those of curcumin plus $H_2O_2$ ($17.1{\pm}0.8\;nmol/L{\times}10^6$) or $H_2O_2$ group ($22.5{\pm}1.9\;nmol/L{\times}10^6$) from 3 to 9 hr incubation periods. The DF by sperm chromatin dispersion (SCD) test and ROS production measured by 2',7'-dichlorofluorescein (DCF) fluorescence intensity were no significantly difference through all experimental groups (p>0.05). Correlation among evaluation methods for sperm quality, motility vs MI and DF vs ROS was positively correlated while motility vs DF and ROS vs LPO were negatively correlated in all treatment groups. These results demonstrate that curcumin can effectively improve the sperm quality during in vitro storage of boar semen through its hydrogen peroxide scavenging mechanism as an antioxidant.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.167-172
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• 2004

This study was performed to examine the correlations among dog sperm viabilities evaluated by flow cytometry, by microscopic evaluation (ME), by carbo-xifluorescein diacetate and propidium iodide (CFDA/PI) and by hypoosmotic swelling (HOS) test. Semen were collected from 5 dogs ranging in age from 2 to 4 years. Each ejaculate was divided into 3 aliquots and different proportions of freeze-killed cells were added to each aliquot (1:0, 1:1 and 1:3). In the other experiment, semen was extended with Sweden extender containing 5% glycerol and equex STM paste, and frozen using liquid nitrogen vapor. Fresh and frozen-thawed dog sperm viability were assessed by flow cytometry using PI staining method. The accuracy of flow cytometry was evaluated by comparing with other classic assessments, microscopic evaluation, epifluorescence microscopic analysis using CFDA/PI, and HOS test. High correlations of sperm viabilities were found among flow cytometry, epifluorescence evaluation, HOS test (p<0.01) in fresh semen. Especially, sperm viability assessed by HOS test was highly correlated with viability by flow cytometry in all the ratios of live and dead spermatozoa, 1:0, 1:1 and 1:3 (p<0.01). The viability evaluated by ME were significantly correlated with that by flow cytometry in ratios of 1:0 and 1:3 (p<0.05) however, there was no significance in ratio of 1:1. The viability evaluated by C/p were highly correlated with that by flow cytometry in ratio of 1:0 and 1:1 (p<0.01) and significantly correlated in ratio of 1:3 (p<0.05). In frozen-thawed spermatozoa, the viability determined by HOS test was considerably correlated with that by flow cytometry (p<0.01). There was significant correlation between the viabilities by ME and by flow cytometry (p<0.05). But the viability evaluated by CFDA/PI was not correlated with viability by flow cytometry. The result from this study validate the use of flow cytometry as a precise method for assessing the viability of fresh and frozen-thawed dog spermatozoa.

• Journal of Environmental Health Sciences
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• v.44 no.2
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• pp.153-159
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• 2018

Objectives: This study was carried out to determine $LD_{50}$ of benzo[a]pyrene to decide the possibility to designate them as toxic substance on the Act on the Registration and Evaluation, etc. of Chemical Substances, and to suggest that they should be managed in what level on the Chemical Control Act. Methods: Based on the result of a preliminary study, 300 mg/kg was set as the middle dose. A highest dose of 2,000 mg/kg and a lowest dose of 50 mg/kg were selected based on the OECD TG 423. Benzo[a]pyrene was orally administered once to female and male SD rats at dose levels of 50, 300, 2,000 mg/kg (body weight). All animals were monitored daily for clinical signs and mortality over 14 days. Also testicular spermatid count, motility and etc. were examined as well. Results: Under the condition of this experiment, $LD_{50}$ of benzo[a]pyrene was assumed to be >2,000 mg/kg. In the lesion according to autopsy, there were no specific symptoms in the control and experimental groups. At 2,000 mg/kg, a decrease in the sperm motility was observed. Benzo[a]pyrene should be designated to be toxic substance as the material assumed to be reproduction-toxicity on the Act on the Registration and Evaluation, etc. of Chemicals. Therefore we should abide by legal procedures determined by Chemicals Control Act in treating it. Conclusion: Considering the significant result that sperm motility in the experimental group was inferior to that in the reference group, we suggest that benzo[a]pyrene be designated as a toxic substance.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.207-213
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• 2017

Objective: This study investigated the prevalence of infections with human papillomavirus, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium in the semen of Korean infertile couples and their associations with sperm quality. Methods: Semen specimens were collected from 400 men who underwent a fertility evaluation. Infection with above five pathogens was assessed in each specimen. Sperm quality was compared in the pathogen-infected group and the non-infected group. Results: The infection rates of human papillomavirus, C. trachomatis, U. urealyticum, M. hominis, and M. genitalium in the study subjects were 1.57%, 0.79%, 16.80%, 4.46%, and 1.31%, respectively. The rate of morphological normality in the U. urealyticum-infected group was significantly lower than in those not infected with U. urealyticum. In a subgroup analysis of normozoospermic samples, the semen volume and the total sperm count in the pathogen-infected group were significantly lower than in the non-infected group. Conclusion: Our results suggest that infection with U. urealyticum alone and any of the five sexually transmitted infections are likely to affect sperm morphology and semen volume, respectively.

• Toxicological Research
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• v.33 no.3
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• pp.255-263
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• 2017

Chemotherapy is associated with male infertility. Cisplatin (cis-diamminedichloro-platinum (II) (CDDP) as a chemotherapy medication used to treat a number of cancers has been reported to most likely induce testicular toxicity. Administration of antioxidants, such as pentoxifylline (PTX) may reduce some Adverse Drug Reactions (ADRs) of CDDP. Therefore, this study investigated the potentially protective effects of PTX on CDDP-induced testicular toxicity in adult male rats. For this purpose, 42 male rats were randomly divided into 7 groups. The rats were orally pretreated with PTX at the 3 doses of 75, 150, and 300 mg/kg once a day for 14 successive days. On the $14^{th}$ day of the study, they were intraperitoneally (IP) administered with a single dose of CDDP (7 mg/kg). Finally, the sperm/testis parameters, serum levels of reproductive hormones, including testosterone, Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH) as the pivotal endocrine factors controlling testicular functions, and histopathological changes of testis tissue were examined. Pretreatment with the two doses of 75 and 150 mg/kg PTX indicated significant increases in the sperm count and motility induced by CDDP administration. The right and significantly left testis weights were decreased following the treatment with 300 mg/kg of PTX plus CDDP. However, 75 mg/kg of PTX plus CDDP showed the best near-to-normal histopathological features. The results demonstrated that PTX alone enhanced some parameters, such as the sperm count, while reducing other parameters, including sperm fast motility and germ layer thickness. Furthermore, despite testosterone or LH levels, the mean serum FSH level was significantly augmented by the doses of 75 and 150 mg/kg. It was concluded that PTX administration cannot reduce CDDP-induced testicular toxicity even at high doses (e.g., 300 mg/kg), while it seemed to partially intensify CDDP toxicity effects at a dose of 75 mg/kg. Thus, further research is required in this regard.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.387-396
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• 2005

The aims of the study were to establish a short-term screening test for detecting testicular toxicity of chemicals in rats and to determine whether a 2-week administration period is sufficient to detect testicular toxicity of 2-bromopropane (2-BP) as an example. Male Sprague-Dawley rats were subcutaneously administered with 1000 mg/kg/day of 2-BP or its vehicle for 2 weeks. Ten male rats each were sacrificed on days 3, 7 and 14 after the initiation of treatment. Parameters of testicular toxicity included genital organ weights, testicular sperm head counts, epididymal sperm counts, motility and morphology, and qualitative and quantitative histopathologic examinations. The early histopathological changes observed on day 3 of treatment included degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, and decreased number of spermatogonia in stages II and V. On day 7 of treatment, atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in stages VII and XII. On day 14 after treatment, a significant decrease in the weights of testes and seminal vesicles was found. Atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in all spermatogenic stages were also observed. In addition, a slight non-significant decrease in testicular sperm head counts, daily sperm production rate and epididymal sperm counts was found. The results showed that 2 weeks of treatment is sufficient to detect the adverse effects of 2-BP on male reproductive organs. It is considered that the short-term testicular toxicity study established in this study can be a useful tool for screening the testicular toxic potential of new drug candidates in rats.