• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.31-41
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• 1994

Morphological estimation of human spermatozoa is complicated by the fact that there is great natural variation in shape. This natural variation in shapes makes it difficult to say which forms are associated with infertility and which are normal variations. Possibly post coital test or in vitro cervical mucus penetration tests will help to clarify this question by showing which sperm are capable of penetration. The purpose of this investigation was performed to assess distribution of various morphological abnormalities according to the ability of sperm to penetrate cervical mucus. The sperm-mucus penetration using hen's egg white as substituting mucus for human cervical mucus was done in 45 fertile men with normal semen analysis and 122 infertile men with abnormal seminal parameters more than one. The female partners of 122 infertile couples showed normal results in the female fundamental test for fertility. Conventional semen analysis was evaluated according to the WHO standard normal(l980). The detailed classification of the abnormal sperm was made according to David et al(l975). The vitality of the sperm samples determined by eosin yellow-nigrosin stainig according to the method of Eliasson(l977). Results were as follw; 1. The patients had significantly lower total sperm count, motility (%), normal morphology (%), viability and total functional sperm fractions(TFSF) than fertile donors. 2. The mean value of sperm penetration distance of the patients(28.69${\pm}$11.02mm) showed significantly lower than fertile donors(37.33${\pm}$5.49mm). And 43/45 fertile donors(95.5%) as well as 57/122 patients(46.7%) had over 30mm in sperm penetration distance respectively. While 2/45 fertile donors(4.5 %) and 65/122 patient(53.3%) had under 30mm in sperm penetration distance respectively. 3. The morphological abnormalities in fertile donors were significantly lower 23.04${\pm}$5.83% (head = 12.89${\pm}$4.98, neck=6.11${\pm}$3.83%, and tail=3.43${\pm}$2.65%), compared to 36.03${\pm}$14. 40% in patients(head = 15.98 8.60%, neck 11.20${\pm}$6.56% and tail=8.70${\pm}$6.55%). Also, 3 types of sperm abnormalities including head, neck and tail were significantly lower in patient than fertile donors, respectively. Both the patients and fertile donors showed higher distribution of sperm with abnormal head than abnormal neck and tail. 4. The mean morphological abnormalities(SP>30mm) of the patients(30.68 11.64%; head = 15.95${\pm}$9.35%, neck=8.14${\pm}$4.21 %, tail=6.56${\pm}$5.64%) were significantly lower compared to patients(40.72${\pm}$15.01 %; head=16.02${\pm}$7.69%, neck 13.89${\pm}$7.82%, tail=1O.58${\pm}$6.75%) under 30mm in sperm penetration distance. Also, both groups over 30mm and under 30mm in sperm penetration showed distance higher distribution of sperm with abnormal head than abnormal neck and tail. The morphological abnormalities of head did not show significant difference but abnormal neck and tail were significant difference between the over 30mm and under 30mm group in sperm penetration distance.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.97-102
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• 2013

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under $30^{\circ}C$ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in $0.3^{\circ}C$. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group ($43.3{\pm}4.7%$) was significantly (p<0.05) higher than other treatment groups (Tissue: $16.3{\pm}2.7%$ and Water: $27.5{\pm}3.1%$), dying sperm (SYBR+/PI+) in Air treatment group ($55.6{\pm}4.7%$) was significantly lower than other treatment groups (Tissue: $77.6{\pm}3.2%$ and Water: $67.6{\pm}3.3%$) (p<0.05). Acrosome reaction in Air treatment group ($0.2{\pm}0.1%$) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: $0.7{\pm}0.2%$ and Water: $0.5{\pm}0.1%$), the acrosome reaction in Air treatment group ($28.6{\pm}2.8%$) within all sperm also was significantly lower than other treatment groups (Tissue: $44.2{\pm}1.8%$ and Water: $36.2{\pm}2.0%$) (p<0.05). And mitochondrial intact in Air treatment group within live ($97.1{\pm}0.4%$) and all ($61.9{\pm}3.3%$) sperm were significantly higher than other treatment groups (Tissue: $85.2{\pm}3.3%$, Water: $87.8{\pm}2.9%$ within live sperm and Tissue: $49.28{\pm}3.7%$, Water: $42.0{\pm}3.1%$ within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1561-1565
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• 2006

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without $250{\mu}M$ $H_2O_2$. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group ($85.1{\pm}0.5$ and $72.4{\pm}0.3$, p<0.05). The sperm motility in taurine with $H_2O_2$ after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with $H_2O_2$ ($75.3{\pm}0.3$ and $69.6{\pm}2.9$, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM ($76.0{\pm}0.6$) and 50 mM taurine groups ($78.0{\pm}0.7$), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (${\geq}48%$ in 9 h and ${\geq}42%$ in 12 h) compared to controls ($43.0{\pm}2.1$ and $31.0{\pm}0.6$, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics(motility, viability, survival and membrane integrity).

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.181-186
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• 2011

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

• Journal of Veterinary Clinics
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• v.28 no.6
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• pp.571-575
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• 2011

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.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.21-29
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• 1994

Subzonal insemination(SUZI) has been proposed for patients with severe male factor and previous fertilization failure. However, very low fertilization rates still persisted. The aims of this study were firstly, to examine the relationships between the fertilization rate and sperm parmeters, sperm incubation media and time, secondly, to evaluate the outcome of 119 cycles of SUZI applied the modified sperm preparation method. The fertilization rates were influenced more sensitively by sperm preincubation media and time than by sperm parameters. According to preincubation media and time, the fertilization rates were 43.3% in 50% follicular fluid (HFF), 36.6% in 10% fetal cord serum(FCS), and with the time, increased in FCS, but decreased in HFF. In regrd with sperm parameters, the fertilization rates were 42.9% in normal and 37.6% in subnormal group. The best results were obtained from SUZI by the spermatozoa incubated in 50% HFF for 6-8 hours. So we tried 119 cycles of SUZI(normal; 39 cycles, subnormal; 80 cycles) using the preparation method of 6-8 hour incubation in 50% HFF. There were no signigicant differences in the fertilization rates between normal(125/269, 46.4%) and subnormal sperm(264/635, 41.6%). Contrary to the fertilization rates, pregnancy outcomes were different between both groups. Better results obtained from the subnormal group than the normal in the number of transferred embryos, that of good embryos, and developmental rate of the fertilized eggs. The pregnancy rates per transfer were totally 13.3%(13/98),20.0%(13/65) in subnormal group. In the normal group, 2 patients showed ${\beta}$-hCG positive, but resulted in chemical pregnancy. Of 13 clinical pregnancies, two aborted, 6 on-going, and 5 delivered. In conclusion, SUZI is an effective technique to overcome fertilization failure for male factor and unexplained. The fertilization rate is influenced by sperm parameters, sperm incubation media and time. Also the quality of oocytes might be important for pregnancy as same as that of sperm.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.107-115
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• 1993

Procedures to separate motile. normal & motile and acrosome-reacted sperm with high efficiency have clinical application in Assisted Reproductive Technology in terms of increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. This study evaluated the effects of 10 sperm preparation techniques [Swim-up from a washed pellet (SU). Swim-up from semen (SO). Continuous Percoll Gradients I (PIC). Discontinuous Percoll Gradients I (PID). Continuous Percoll Gradients II(P II C). Discontinuous Percoll Gradients II(P II D), SpermPrep (SFC). Wang's tube (WT). Albumin Gradients (AG), Low temperature capacitation (LTC)] on motility (%), normal morphology (%), motile sperm recovery rate(%). morphologically normal & motile sperm recovery rate (%), true acrosome reaction (%) and fertilizing ability. A P II D proved to be an effective means of separating morphologically normal & motile sperm. Our results indicated the P II D has advantages as compared with other methods in terms of recovery rate. enhancement of motility and normal morphology. And a LTC seems to be an effective means of enhancing the true acrosome reaction and fertilizing ability. These results suggest that the combined method of LTC and P II D for separation of morphologically normal & motile sperm and acrosome reacted sperm may be a useful procedure for intrauterine insemination and in vitro fertilization in the management of male factor infertility as well as for isolation of subpopulation of sperm for basic research.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.105-115
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• 2002

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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.69-69
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• 2002

Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.