• 한국가축번식학회지
• /
• 제15권3호
• /
• pp.189-193
• /
• 1991

This study was carried out ot investigate effects of X-537A on hydrogen ion concentration insperm washed solution and sperm acrosome reaction. The results obtained were as follows. 1. When bovine sperm was twice washed with SHP solutions of pH 6.8 and 7.4 and again washed with SHP solution containing 4$\mu$M of X-537A, in case of pH 6.8 the sperm washed with 4$\mu$M of X-537A showed signifciantly(p<0.01) higher hydrogen ion concentration in sperm washed solution than the sperm washed without X-537A. 2. When the sperm was twice washed with SHP solution and then washed with SHP solution containing 4$\mu$M of X-537A, sperm acrosome rection rate was signifciantly(p<0.01) increased from 12min after incubation in the sperm washed without X-537A, but was signifciantly(p<0.01) increased from 8 min after incubation in the sperm washed with 4$\mu$M of X-537A. 3. When the sperm was twice washed with SHP solution and then washed with SHP solution containing 0, 4 and 40$\mu$M of X-537A, and then incubated in m-TALP for 120 min, sperm acrosome reaction rate was significantly(p<0.01) increased from 15 min after incubation in 0, 4 and 40$\mu$m OF X-537A. However at 60 min incubation 40$\mu$M of X-537A showed significantly(p<0.01) higher sperm acrosome reaction rate than 0 and 4$\mu$M and at 120 min incubation 4 and 40$\mu$M of X-537A showed signifciantly(p<0.01) higher acrosome reaction rate than 0$\mu$M of X-537A.

• 한국수정란이식학회지
• /
• 제28권1호
• /
• pp.41-48
• /
• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• 한국패류학회지
• /
• 제20권1호
• /
• pp.17-26
• /
• 2004

In present study, attempts were made to preserve abalone (Haliotis discus hannai) sperm in liquid form at low temperature, to evaluate the effect of various diluents in short-term storage on sperm, and cryopreservation procedures were optimized for the cryoprotectants and freezing rates, as well as the motility, survival rate, and the ultrastructural changes of sperm after short-term storage and cryopreservation were observed. The abalone sperm reached maximum motility until about 4 min after activation. The motility was constant for about 16 min, after which it dropped gradually, and about 50 min later all motility ceased. In Hanks' balanced salt solution (HBSS, 300 and 400 mOsmol/kg) and 150, 250 and 350 mOsmol/k artificial seawater (ASW), the sperm was immotile. After 100% ASW was added, motility of those sperm, which are in 300, 400 mOsmol/kg HBSS, 250, 350 mOsmol/kg ASW, could be again restored incompletely. Sperm motility can be maintained for 20 days of cold storage only in ASW of 850 and 1200 mOsmol/kg. A high motility index of 3.5-4.5 was observed for the first 8 days in 850 and 950 mOsmol/kg ASW. In other diluents sperm motility was constant less than 10 days, and the motility index was obviously lower than that of sperm in 850 and 1200 mOsmol/kg ASW. After 20 days of cold storage, survival rates of 10.2%-20.7% were obtained in ASW and 300 mOsmol/kg HBSS, and that in 400 HBSS (65.3%) was significantly higher than others. The constant period of sperm motility stored in 850 mOsmol/kg ASW was obviously longer than that in 1200 mOsmol/kg ASW after 6 days of storage. The sperm plunged into liquid nitrogen all died except that sperm using 15% glycerol as cryoprotectant restored 10.4% of motility. The highest motility index (3.4) was obtained with 5% glycerol and freezing procedure: $-50^{\circ}C$/min from $20^{\circ}C$ to $-80^{\circ}C$.

• 한국동물학회지
• /
• 제38권4호
• /
• pp.514-522
• /
• 1995

생쥐 정자의 수정능력 획득과정 및 첨체반응을 전후로 정자표면과 albumin의 상호작용을 조사하였다. 부정소 정자를 체외에서 배양하는 과정에서 정자표면에서 이탈된 단백질의 분석과 함께 FITC-bovine serum albumin으로 정자를 형광염색하여 정자표면에 대한 albumin의 결합양상의 변화를 조사하였다. 90분간 정자를 배양한 수 정자를 제거한 후 농축한 배양액내에 정자 또는 부정소액에서 기원한 여러종의 단백질가 함께 albumin이 다량으로 발견되었다 정자의 체외배양 과정에서 일어나는 albumin의 이탈은 배양액내의 $Ca^2$+과 무관하게 일어났다. BSA-FITC는 정소내 정자의 두부표면에 미약하게 결합한 반면 미부부정소 정자의 첨체표면에는 다량 결합하였다. $Ca^2$+-ionophore인 A23187으로 첨체반응을 유발한 정자의 두부 표면에서는 후첨체부위만이 강하게 염색되었다. 이러한 결과는 정소 및 웅성 생식수관니에서 정자표면에 부착된 albumin이 자성 생식수관을 거치는 동안 이탈됨을 시사하며 이러한 현상은 정자의 수정능력획득과 밀접한 관련이 있는 것으로 사료된다.

• 한국임상수의학회지
• /
• 제10권1호
• /
• pp.1-10
• /
• 1993

To investigate the availability of hamster test in assaying the fertilizing capacity of dog sperm and the effect of canine sperm motility on sperm binding and penetration, semen were collected from four dogs(three dogs had been proven to be fertile and one dog to be subfertile during the past two years) and then preserved in BWW(Biggers, Whitten, Whittingham) medium for about 20 hours. The semen were given each different treatment according to the experimental design and coincubated with zona-free hamster ova for 5 hours. The ova were stained by lacmoid and examined under phase contrast microscope to investigate the rates of ova bound with sperm(sperm binding) and ova penetrated by sperm(penetration), and also numbers of both bound and penetrated sperm per ovum. In comparison between fertile dogs and a subfertile dog, the rate of sperm binding was higher in fertile dogs than the subfertile dog(p<0.01, p<0.05). The number of bound sperm per ovum was considerably higher in a fertile dog than the subfertile dog((p<0.01), and also difference of number of the bound sperm was obtained among the fertile dogs(p<0.01, p<0.05). The rate of penetration as well as the number of penetrated sperm per ovum was higher in the fertile dogs than the subfertile dog(p<0.01, p<0.05). In fertile dogs. the canine semen preserved at 4$^{\circ}C$ for 18 to 22 hours showed from 30 to 80% motility at Insemination, however, no difference in hamster test was obtained according to different degree of sperm motility. These results indicated that hamster test would be of avail in assaying the fertilizing capacity of dog sperm.

• 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.

• Clinical and Experimental Reproductive Medicine
• /
• 제48권1호
• /
• pp.61-68
• /
• 2021

Objective: This study was conducted to investigate the relationship of semen parameters in samples used for intracytoplasmic sperm injection (ICSI) with fertilization and pregnancy rates in infertile couples. Methods: In this prospective study of Infertile couples with male factor infertility that had undergone ICSI, fractions of the same semen samples obtained for microinjection (to ensure the best predictability) were evaluated to determine the semen parameters and sperm DNA fragmentation index (DFI) on the day of oocyte recovery. Results: In total, 120 couples completed the study and were subdivided into fertilized (n=87) and non-fertilized couples (n=33). The fertilized couples were further classified into pregnant (n=48) and non-pregnant (n=39) couples. Compared to non-fertilized and non-pregnant couples, fertilized and pregnant couples showed statistically significantly higher sperm viability and percentage of normal sperm morphology, as well as significantly lower sperm DFI values. A receiver operating characteristic curve analysis of data from the 120 ICSI cycles showed that sperm viability, normal sperm morphology percentages, and sperm DFI were significant prognostic indicators of fertilization at cutoff values of 40%, 7%, and 46%, respectively. A sperm DFI of 46% showed sensitivity and specificity of 95% and 90%, respectively, for predicting fertilization, and no clinical pregnancies occurred in couples with a sperm DFI above 46%. Conclusion: Semen parameters from the ICSI day sample, especially sperm viability, normal morphology, and DFI, had an impact on fertilization and pregnancy outcomes in ICSI cycles.

• 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.

• Clinical and Experimental Reproductive Medicine
• /
• 제49권1호
• /
• pp.40-48
• /
• 2022

Objective: As the associations of sperm DNA fragmentation with morphology have not been examined in detail, this study aimed to investigate the relationship between abnormalities of morphological details and DNA integrity in human sperm. Methods: In this cross-sectional study, men from infertile couples were enrolled at Hue Center for Reproductive Endocrinology and Infertility, Vietnam. Conventional semen parameters, including morphological details, were analyzed following the World Health Organization 2010 criteria. Sperm DNA fragmentation was evaluated using a sperm chromatin dispersion assay. The relationships and correlations between semen parameters, sperm morphology, and the type of halosperm and the DNA fragmentation index (DFI) were analyzed. Results: Among 130 men in infertile couples, statistically significant differences were not found in the sperm halo type between the normal and abnormal sperm morphology groups. The percentage of round-head spermatozoa was higher in the DFI >15% group (16.98%±12.50%) than in the DFI ≤15% group (13.13% ±8.82%), higher values for amorphous heads were found in the DFI >15% group, and lower values for tapered heads were observed in the DFI ≤15% group; however, these differences were not statistically significant. Small-halo sperm and the DFI were positively correlated with round-head sperm (r=0.243, p=0.005 and r=0.197, p=0.025, respectively). Conclusion: The rate of general sperm morphological abnormalities in semen analysis was not related to sperm DNA integrity. However, round sperm heads were closely associated with sperm DNA fragmentation.

• Clinical and Experimental Reproductive Medicine
• /
• 제50권4호
• /
• pp.262-269
• /
• 2023

Objective: The aim of this study was to compare semen parameters and sperm DNA fragmentation (SDF) and explore the relationship between semen parameters and SDF between 2 and 7 days of abstinence and a short abstinence period (within 4 hours) in oligozoospermic infertile patients. Methods: Two semen samples were collected from infertile oligozoospermic men (n=34) after an abstinence period of 2 to 7 days and within 4 hours, respectively. Sperm parameters were compared between the two abstinence duration groups, including semen volume, sperm concentration, total sperm count, sperm motility, total motile sperm count (TMSC), morphology, and SDF. Results: The semen volume, concentration, and total sperm count were significantly decreased after 4 hours of abstinence than after 2 to 7 days of abstinence, with median differences of 1.2 mL (p<0.001), 2×10⁶/mL (p=0.011), and 9.6×10⁶/ejaculation (p<0.001), respectively. TMSC was significantly lower after a short abstinence, with a median difference of 4.24×10⁶/ejaculate (p<0.001). However, there were no significance differences in the percentage of motility, the SDF, and the percentage of sperm with normal morphology. Interestingly, volume, concentration, total sperm count, sperm motility, and SDF, but not TMSC, exhibited significant linear correlations between the two abstinence groups in univariate regression analysis, except for TMSC. Conclusion: In oligozoospermic men, the volume, concentration, and total sperm count were significantly lower after a short abstinence period, but without adverse effects on sperm motility and SDF.