• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.339-344
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• 2000

Objective: To evaluate silane-coated silica particles (Sil-select) as an alternative to polyvinylpyrrolidone-coated particles (Percoll) for gradient separation of spermatozoa, for use in assisted reproduction. Methods: 20 normal semen based on WHO criteria were included in this study. Recovery of motile and morphologically normal spermatozoa after using two-layer Percoll and Sil-select gradient respectively was recorded. Motility, HOST (hypoosmotic swelling test) and the detection of malondialdehyde for LPO (lipid peroxidation) after 24 h of incubation at $37^{\circ}C$ in a 5% $CO_2$ incubator were compared. Results: Percoll (78.5%) and Sil-select (79.1%) showed a significant increase in the motility compared to ejaculate (60.9%) but no difference between Percoll and Sil-select. Normal sperm morphology significantly increased after Percoll (57.6%) and Sil-select (53.7%) compared to ejaculate (35.8%) but no difference between Percoll and Sil-select. No differences in the recovery of motile spermatozoa and motility, HOST and the production of malondialdehyde after 24 h incubation were found when comparing the use of Percoll and Sil-select. Conclusion: Sil-select seems to be an attractive alternative to Percoll for sperm separation in assisted reproduction.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.4
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• pp.193-198
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• 2016

Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. G6PD plays a key role in the pentose phosphate pathway, which is a major source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH provides the reducing equivalents for oxidation-reduction reductions involved in protecting against the toxicity of reactive oxygen species such as $H_2O_2$. We hypothesized that G6PD deficiency may reduce the amount of NADPH in sperms, thereby inhibiting the detoxification of $H_2O_2$, which could potentially affect their motility and viability, resulting in an increased susceptibility to infertility. Methods: Semen samples were obtained from four males with G6PD deficiency and eight healthy males as a control. In both groups, motile sperms were isolated from the seminal fluid and incubated with 0, 10, 20, 40, 60, 80, and $120{\mu}M$ concentrations of $H_2O_2$. After 1 hour incubation at $37^{\circ}C$, sperms were evaluated for motility and viability. Results: Incubation of sperms with 10 and $20{\mu}M\;H_2O_2$ led to very little decrease in motility and viability, but motility decreased notably in both groups in 40, 60, and $80{\mu}M\;H_2O_2$, and viability decreased in both groups in 40, 60, 80, and $120{\mu}M\;H_2O_2$. However, no statistically significant differences were found between the G6PD-deficient group and controls. Conclusion: G6PD deficiency does not increase the susceptibility of sperm to oxidative stress induced by $H_2O_2$, and the reducing equivalents necessary for protection against $H_2O_2$ are most likely produced by other pathways. Therefore, G6PD deficiency cannot be considered as major risk factor for male infertility.

• Journal of Aquaculture
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• v.8 no.3
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• pp.149-157
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• 1995

In order to obtain the basic knowledges concerned to the semen preservation of aquacultural fishes, studies on the physical and chemical properties of semen, and sperm motility with the different osmotic pressures making by adding $Na^+,\;K^+,\; Mg^{++},\;and\;Ca^{++}$ to artificial seawater (ASW) were conducted in black seabream, Acanthopagrus schlegeli. Average semen volume per fish in one strip was 1.97ml and sperm concentration was $2.33\pm1.30\times10^{10}$ sperm/ml. Spermatocrit and pH of semen were $90.6\pm5.0\;and\;8.3\pm0.1$, respectively, Osmotic pressures of rearing seawater, seminal fluid and plasma were $939\pm24,382\pm70\;and\;342\pm77$ mOsm/l, and $Na^+,\;K^+$ and $Cl^-$ concentrations of seminal fluid were $169.5\pm4.5,\;4.9\pm2.2,\;156.0\pm2.0\;mM/l$, respectively. When semen were diluted by using $Na^+,\;K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, only $Na^+$ free ASW had no sperm motility. As raising osmotic pressure graduary by addition of 1M NaCl to the $Na^+$ free ASW, spermatozoa showed the high motilities in 457-1128 mOsm/l, but the low motilities in 1398-1736 mOsm/l. In the case of same treatments with 1M of KCl, $MgC1_2\;and\;CaC1_2$ to the $K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, spermatozoa revealed the high motilities in $904\~1434,\;818\~1175\;and\;956\~1343$ mOsm/l, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.393-403
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• 2000

Objective: To investigate the effects of ROS on kinematic parameters in human spermatozoa. To verify the changes in above parameters, human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spemlatozoa were incubated under low $O_2 (5%) condition. Methods: CASA was employed to analyze sperm motion parameters. Results: Under $H_2O_2 treatment, all kinematic parameters of spermatozoa were dramatically increased during 30 min, but gradually decreased thereafter. Under the low concentration of $H_2O_2 (125 ${\mu}M$ and 250 ${\mu}M$), the movement velocity (VAP, VCL, VSL) decreased, but forward movement increased. Under the 1mM $H_2O_2, sperm showed reduced kinematic parameters except during first 30 min of incubation. In the cases of X-XO and SNP treatment, the movement velocity increased but the forward movement reduced. After incubation for 3 hr treatment, the kinematic parameters gradually decreased in high concentration of X-XO. However these parameters maintained or increased in low concentration of X-XO. There was no obvious changes in the above parameters in the high concentration of SNP. In the presence of high concentration of lymphocytes, all parameters decreased. Under the 5% $O_2 condition, the parameters of the movement velocity and movement pattern increased, but forward movement decreased. Taken togethers, it suggested that ROS increased the movement velocity but decreased the forward movement and lateral head replacement. $H_2O_2, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within I h of incubation. There was no significant difference in capacitation between low- and high $O_2 group. Conclusion: The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation. These results demonstrate that low concentration ROS facilitates the movement velocity but high concentration ROS was inhibitory.

• Journal of Veterinary Clinics
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• v.16 no.2
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• pp.375-380
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• 1999

This study was conducted to develop an intrauterine inseminator (IUI) to deposit of frozen semen into uterus and to evaluate the results obtained after artificial insemination by IUI. Two Japanese spitzs (2 to 4 years of age) were used as semen donors. Semen was collected by manual masturbation into sterile glass collection tubes and separated into 3 fractions with only the sperm-rich fractions retained for further examination. Sperm motility >70%, sperm concentration of 200 to $400{\times}10^6 cells/ml$$\times$g for 5 min and poured out the suspended solution, and then diluted with 2 ml Tris-buffer which was consisted of 2.4 g Tris, 1.4 g citric acid, 0.8 g glucose, 0.1 $\mu\textrm{g}$/ml streptomycin, 100 IU/ml penicillin, 20 ml egg yolk to 100 ml mili-Q water (Ext I) or supplemented with 8 ml glycerol and 1 ml Equex STM paste to 100 rnl (Ext II). The diluted semen was cooled to 5$^{\circ}C$ in cold room, where the temperature in the sample reached 5$^{\circ}C$. Two h after beginning the cooling procedure, 2 ml of Ext II, also at 5$^{\circ}C$, was added and mixed by gently reversing the tubes several times during 1 h. The final sperm concentration for freezing was approximately $50{\times}10^6 cells/ml$. After equilibration, the semen was loaded into 0.5 ml straw and frozen on the liquid nitrogen vapour in styrofoam box. The straws were thawed at 7$0^{\circ}C$ for precisely 6 sec. After thawing of each straw, the frozen semen can survived over 50% motility. All the females were inseminated twice with 1 ml of $25{\times}10^6 cells/ml$

• Clinical and Experimental Reproductive Medicine
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• v.10 no.1
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• pp.7-24
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• 1983

On the basis of the semen analysis in 66 subjects, they were divided into six different groups: Group I consisted of 16 normal subjects with sperm counts of over 40 ${\times}10^6$/ml and motility of over 40 percent, Group II, 7 subjects with normal sperm counts, but motility of under 40 percent, Group III, 15 oligospermic patients with under 40 ${\times}10^6$/ml, Group IV 14 azoospermic patients, Group V, 10 patients with vasectomy and Group VI, 4 abnormal patients with 2 cases of hypoplastic testis, 1 case of Klinefelter's syndrome and 1 case of testis tumor. After seperation of semen into sperm and seminal plasma by centrifugation, the protein contents and the activities of hyaluronidase, ${\beta}$-N acetylglucosaminidase, ${\beta}$-glucuronidase, arylsulfatase, acrosin and azocoll proteinase in seminal plasma were measured. Vasectomy group has 30 percent less of total protein than normal group. For the comparison of enzyme activities of seminal plasma, it could be assumed that the enzymes in seminal plasma were not contaminated with the enzymes of spermatozoa by testing the enzymes of the seminal plasma from the vasectomy and azoospermic groups. It had been reported that hyaluronidase was only released from spermatozoa, however, the result obtained in this investigation showed that azoospermic and vasectomy group had high specific activities of hyaluronidase. The results indicated that hyaluronidase was not only from the testis but also from the male accessory sexual glands. Oligospermic group (Group III) showed the lowest total activity of hyaluronidase among them. The specific activities of ${\beta}$ -N-acetylglucosaminidase was high in oligospermic group (Group III) and low in vasectomy group (Group V). These results were contradictory with the pattern of hyaluronidase activities. This indicated that the spermatozoa which were stayed in epididymis would increase the activity of this enzyme. The specific activity of ${\beta}$ - glucuronidase was low in oligospermic and vasectomy groups. Group VI including testis tumor had remarkably high arylsulfatase activity. Arylsulfatase, a typical lysosomal enzyme, has been known to be released unusually large amounts from certain tumor cells. Arylsulfatase was also released with high activities from azoospermic and vascetomy group. This result indicated that this enzyme was also released from the sources other than testis. Acrosin, a proteolytic enzyme locating in the sperm acrosome, was not found throughout all the samples of seminal plasma. The activities of azocoll proteinase, a non-specific neutral proteinase was nearly identical in all the groups. This enzyme must have been released from the sources other than testis.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.31-39
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• 2013

Alpha-tocopherol as an antioxidant acts in preservation of chilled semen by preserving cell membrane damage from lipid peroxidation. Optimum concentrations of ${\alpha}$-tocopherol in egg yolk-citrate (EYC) extender need to be studied in crossbred bull's semen. Different concentrations of ${\alpha}$-tocopherol viz. 0, 1, 2, 4 and 6mg per ml of extender were used. Semen was collected once a week from four bulls used to regular collection, aged 4 to 7 years, weighing 320 to 450 kg, and with body condition score 4 to 4.5 and scrotal circumference 23 to 32 cm. Semen was evaluated routinely and sperm morphology was viewed under light microscope at ${\times}1,000$ magnification after fixing with buffered formal saline. Over 90% had normal head, acrosome, mid-piece and tail. Semen was diluted with egg-yolk-citrate extender to produce $15{\times}10^6$ spermatozoa/ml and 0, 1, 2, 4 and 6 mg/ml ${\alpha}$-tocopherol were added. The semens amples were kept at $8^{\circ}C$. Sperm motility and viability were examined daily up to 5 days under light microscopy at ${\times}200$ magnification. Sperm viability was acceptable (${\geq}40%$) up to the $4^{th}$ day with all concentrations of ${\alpha}$-tocopherol and up to the $5^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. Sperm motility was acceptable (${\geq}40%$) up to the $3^{rd}$ day irrespective of ${\alpha}$-tocopherol concentration, and up to the $4^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. It is suggested that the lifespan of chilled semen may be extended up to 4 days by adding 2mg/ml ${\alpha}$-tocopherol.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1107-1112
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• 2009

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

• Journal of Oriental Medical Thermology
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• v.4 no.1
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• pp.23-28
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• 2005

Purpose : To examine the relation between male infertility and cold hypersensitivity on lower abdomen. Methods : From 2004, 4 to 2005, 10, a total 60(infertile male 30, normal male on semen analysis 30) patients who visited the Oriental gynecological department of Conmaul Oriental Medicine hospital was selected, and their medical records were reviewed retrospectively. We compared the temperature of 3 points(CV17, CV12, CV4) of both group by DITI(Dorex, DITI-16UTI and DITI-Spectrum 9000 MB). Results and Conclusion : The general characteristics such as age, weight, height, BMI, amount and frequency of drinking and smoking of both group were not different statistically. The mean ${\Delta}T1$ between upper and lower abdomen in infertile group was higer than in normal group but they were not different statistically. The mean ${\Delta}T2$ between chest and lower abdomen was $0.46{\pm}0.41$ in infertile group, and $0.18{\pm}0.56$ in normal group, and it shows significant difference statistically. There was no significant relationship between sperm motility and ${\Delta}T1$, ${\Delta}T2$ in infertile group. In this study, we suggest that an inclination of cold hypersensitivity of lower abdomen of infertile male, but it is expressed when it is contrasted to chest of the body rather than upper abdomen.

• Toxicological Research
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• v.21 no.4
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• pp.309-318
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• 2005

Rhus verniciflua Stokes (RVS) has been used as a food supplement and a traditional herbal medicine. In this study, we prepared various flavonoid fractions (RS, RW1, RW2 and RWE) from a hot water extract of RVS and their influence on male reproductive organs and spermatogenesis were studied in rats which were orally administered 200 mg/kg of them for 8 weeks. All experimental groups did not show any significant changes in body weight and blood clinical chemistry for liver function. Plasma testosterone level was elevated about 3.7, 5.2 and 6.3 folds in RW1, RW2 and RWE groups, respectively. The weights of testes and epididymides tended to increase slightly without the statistical significance in RW2 and RWE. The spermatozoon motility and epididymal sperm concentration were significantly increased (P<0.05) in RWE and RW1, respectively, when compared to the control group. There was no significant difference in histology and apparent shape of testes and epididymides among the control and the experimental groups. Collectively, RWE showed effectively the elevation of plasma testosterone level, spermatozoon motility and the epididymal sperm concentration without the significant increase of testis and epidiymides weights. When the component HPLC profile among the flavonoids fractions of RVS was compared, the ratio of components were only different. These findings suggest that the Rhus flavonoid fraction, particularly RWE, can stimulate the androgen-dependent male sexual function and it can be applied to the material of functional food for enhancing the sexual function.