• Journal of Embryo Transfer
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• v.15 no.1
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• pp.103-108
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• 2000

The object of this study was to find simple and effective methods for the speculation of vitality and scorsome status of bovine spermatozoa. The eosin-nigrosin staining, trypan blue staining, and naphthol yellow S-erythrosin B staining was ofter used for the speculation of vitality and/or acrosome status of bovine spermatozoa, respectively. This study has shown that the combined trypan blue-naphthol yellow S-erythrosin B staining is more accurate and effective for the examination of acrosome status and vitality of bovine spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.5
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• pp.415-418
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• 1995

Semen characteristics were examined to find the deterioration of the percentage of live spermatozoa with intact acrosome during hot season using 5 Holstein bulls located in Shirnizu-cho Hokkaido Japan. Spermatozoal viability and acrosomal status were observed simultaneously with triple-stain technique for each spermatozoon. Spermatozoa were divided in four categories (live spermatozoa with intact acrosome, live spermatozoa without intact acrosome, dead spermatozoa with intact acrosome and dead spermatozoa without intact acrosome). Bull and collection month had significant effects on semen characteristics (p < 0.01). The percentage of live spermatozoa with intact acrosome and the percentage of live spermatozoa had the lowest least squares mean by collection month in August (72.7% and 76.7%). These two characteristics indicated the obvious deterioration during hot season. But the fluctuation of these two characteristics were not parallel and the differences between the two characteristics were largest during July to September. The present results indicate the necessity for the simultaneous determination of viability and acrosomal status of each Holstein bull's spermatozoa in order to keep fertility above an acceptable minimum level during hot season.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.3
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• pp.335-340
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• 2006

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury ($HgCl_2$) at 50 to $550{\mu}M$ concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at $550{\mu}M$ mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.81-86
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• 1996

The assessment of acrosomal status is important in evaluating the ability of sperm to fertilize the egg. The acrosomal status of sperm from 47 normal volunteers with proven fertility and 167 subfertile men with not to achieve pregnancy for at least 1 year were evaluated with Acrobeads test(FUSO Pharmaceutical Industries, Ltd, Japan) using immunobeads coated with MH61 monoclonal antibody, which is specific for acrosome-reacted sperm. The mean${\pm}$SD of acrobeads score in 47 volunteer group was $2.8{\pm}0.7$, of which 46(97.9%)cases were ${\geq}$ 2. The mean${\pm}$SD of acrobeads score in 167 subfertile group was $1.7{\pm}0.8$, of which 73(79.3%)cases were ${\leq}$ 1. The aerobe ads score in subfertile group were significantly lower(r=0.294, p<0.05) than those in volunteer group. In subfertile group, acrobeads score were well correlated with the sperm density and motility(r=0.275, r=0.281, p<0.01), but not with semen volume(r=0.16) and serum hormone level(FSH r=0.084, LH r=0.036, testosterone r=0.058, prolactin r=0.006 and estradiol r=0.060)(p>0.05). Of 63 subfertile cases with normozoospermia, 22(34.9%)cases showed 0 or 1 of acrobeads score, which means to accompany with a functional defect in spite of normal morphology. As a results, Acrobeads test is not only a technically simple sensitive procedure with good reproducibility in evaluating the sperm fertilizing capacity but also an useful in the evaluation of effectiveness in the treatment of infertility and the separation of acrosome-reacted sperm in the assisted reproductive technique.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.43-50
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• 1998

This study was performed to determine the effects of nitric oxide on human sperm cell function. Semen samples were obtained from normal healthy volunteers. Motile spermatozoas collected by swim-up method were incubated up to 24 hours in Ham's F-10 medium supplemented with a various concentration of sodium nitroprusside (nitric oxide releasing agent). Sperm motility, hyperactivation, acrosome reaction rate, and acrosin activity were determined. The results are as follows; 1. 1mM of SNP resulted in a significant decrease in sperm motility ($44.8%{\pm}8.9%:78.1%{\pm}6.3%$, and hyperactivation $(10.4%{\pm}6.4%:47.7%:{\pm}9.5%)$ after incubation for 3 hours compared with the control group (Ham's F-10 alone), but had no effect on acrosome reaction. 2. At $100{\mu}M$ SNP, sperm motility was reduced after incubation for 6 hours $(54.8%{\pm}3.2%)$ compared with that of the control group $(82.7%{\pm}8.9%)$, but hyperactivation and acrosome reaction were not affected. 3. However, a lower concentration (less than $10{\mu}M$) of SNP had no effect on sperm motility and hyperactivation for 8 hours of incubation but significantly decreased them when incubation periods were increased up to 24 hours compared with the control group. On the other hand, $1{\mu}M$ and $10{\mu}M$ SNP significantly increased the acrosome reaction rate in both acrosomal status ($17.3%{\pm}5.2%$, $23.5%{\pm}4.7%$, respectively) and acrosin activity ($34.3{\mu}IU{\pm}10.5{\mu}IU,\;45.6{\mu}IU{\pm}5.6{\mu}IU$, respectively) as compared with the control group $(7.0%{\pm}4.0%,\;9.5{\mu}IU{\pm}3.4{\mu}IU)$. These results indicate that SNP, NO releasing agent, has a dose-dependent effects on the sperm cell function. Therefore it may positively affect the fertilization by promoting acrosomal reaction at a lower concentration (less than $10{\mu}M$).

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.265-271
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• 2011

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until $5^{\circ}C$ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.435-439
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• 2011

Sildenafil citrate (SIL) a phosphodiesterase 5 inhibitor (PDE5I) has been used for long time as a first line oral drug for erectile dysfunction. Though it has beneficial effects on erectile organ it also has some adverse effects in other cells and/or tissues related to reproductive system when exposed to longer duration. The objective of the present study is to evaluate the long term effect of SIL on sperm parameters in Wistar albino rat. The animals are divided into two groups, for group I - rats were treated with saline (vehicle alone) and group - II oral administration of 5 mg/kg b.w. of SIL was administrated orally once in a day for 120 days. At the end of the trial period animals were sacrificed and epididymal sperm were subjected to various analysis. Results showed significant reduction in sperm count, motility, viability and morphologically intact sperm in long term PDE5I exposed animals when compared to control. Acrosomal status and fertility test also showed significant reduction in long term PDE5I exposed animals. The present study clearly indicated that long term SIL has shown to induce alteration in sperm quality and quantity, leading to decline in fertility rate. Indicate that SIL impinge on spermatogenesis as well as epididymal function. Understanding the molecular down-stream events involved in long-term exposure to PDE5 inhibitor can be valuable to supervise on related infertility issues and to suggest corrective measures.