• Journal of Veterinary Clinics
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• v.34 no.2
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• Korean Journal of Agricultural Science
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• v.46 no.1
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• pp.195-204
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• 2019

In this study, we examined the effect of a liposome-based extender (Optixcell) and a tris-citric egg-yolk extender (Triladyl) on the frozen-thawed spermatozoa characteristics and the calving rate. The percentages for the total motility of the frozen-thawed spermatozoa were similar in the Optixcell and Triladyl groups. However, among the motile spermatozoa with a straight line velocity (VSL) ${\geq}25{\mu}m/sec$, the curvilinear velocity (VCL, ${\mu}m/sec$), VSL (${\mu}m/sec$), average path velocity (VAP, ${\mu}m/sec$), amplitude of lateral head displacement (ALH, ${\mu}m$), beat cross frequency (BCF, Hz), and plasma membrane integrity of the frozen-thawed spermatozoa for the Optixcell group were significantly higher than those for the Triladyl group. Furthermore, the calving rate in the Optixcell group (79.0%) was higher than that of the Triladyl group (62.8%). However, the acrosomal membrane integrity of the frozen-thawed spermatozoa in the Optixcell and Triladyl groups was not significantly different. These results indicate that semen freezing with Optixcell improved the motility and plasma membrane integrity of frozen-thawed spermatozoa and the calving rate of Hanwoo cows (native Korean cattle). In conclusion, our results suggest that semen freezing with the liposome-based extender Optixcell is more efficient than with the tris-citric egg-yolk extender Triladyl for improved offspring production.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.185-190
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• 2011

To evaluate the effect of spermatozoa culture on glycosidase activity of frozen-thawed spermatozoa in human, the spermatozoa were treated experimentally and assayed for activities of ${\alpha}$-L-fucosidase, ${\alpha}$-D-mannosidase, ${\beta}$-D-galactosidase and N-acetyl-${\beta}$-D-glucosaminidase (${\beta}$-GlcNAc'ase). The ${\beta}$-GlcNAc'ase activity was at least two-folds higher than other glycosidases regardless of spermatozoa incubation (p<0.05). The spermatozoa motility was decreased with incubation periods, but no effects by different glycosidases on the changes of spermatozoa motility during the various periods of incubation. In all glycosidases, the spermatozoa-zona binding rates in spermatozoa without incubation were higher than in spermatozoa incubated for 2 h (p<0.05). ${\beta}$-GlcNAc'ase is present mainly in the plasma membrane of spermatozoa frozen-thawed in human. It was also shown that the glycosidase activity was increased in all glycosidases in spite of lower sperm-zona binding by spermatozoa incubation.

• Asian-Australasian Journal of Animal Sciences
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• v.3 no.2
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• pp.91-96
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• 1990

This experiment was designed to determine whether ${\alpha}$-aminoisobutyric acid (AIB) can be used to predict membrane function of spermatozoa by measuring the uptake of AIB by fresh, stored and frozen-thawed rooster spermatozoa. When spermatozoa were stored at low temperature ($0{\sim}3^{\circ}C$) for 24 h. no difference was found in AIB uptake compared with fresh spermatozoa, whereas storage for 48 h resulted in a slight increase in AIB uptake by spermatozoa. On the one hand, the uptake of AIB by frozen-thawed spermatozoa was less than that by fresh spermatozoa. This suggests possibility of a different membrane transport system between spermatozoa preserved at low temperature ($0{\sim}3^{\circ}C$) and those frozen-thawed. Glycerol used as cryoprotectant may modify rooster sperm membrane in a different manner from cold preservation. Ouabaine ($10^{-4}M$) caused a slight decrease in AIB uptake, but caffeine ($10^{-2}M$) did not influence spermatozoal AIB uptake. These results indicate a successful application of AIB to rooster spermatozoa as a mean for measuring sperm membrane function and suggest a possible alteration of membrane transport system in rooster spermatozoa between cold ($0{\sim}3^{\circ}C$) and cryopreservation ($-196^{\circ}C$).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.43-43
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• 2001

This study evaluated the effect of fertilization-promoting peptide (FPP) on fertilizing ability and glycosidase activity in vitro of spermatozoa frozen-thawed in pig, Using chlortetracycline fluorescence analysis, the various glycosidase analyses and the oocyte penetration test, we have obtained evidence that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. When frozen-thawed spermatozoa was washed with different concentrations of FPP, there were significantly (P<0.05) more acrosome-reacted in medium with 100 nM than 0, 50, 200 and 400 nM. The penetration rates were also highest in medium containing with 100 nM FPP (P<0.05). On the other hand, the $\beta$-N-acetylglucosaminidase activity was at least twofold higher than other glycosidase. In same glycosidase, however, there were no difference in medium with different concentrations of FPP In another experiment, spermatozoa preincubated in medium with or without FPP for 0, 1, 2, 3 and 4 h were inseminated with oocytes matured in vitro. The percentages of spermatozoa that reached acrosome reaction were affected by preincubation and were higher in medium with that than without FPP. When oocytes were inseminated with spermatozoa preincubated in medium with and without FPP during the different periods, however, penetration rates were decreased with preincubation periods of spermatozoa. On the other hand, when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 and 24 h, the penetration rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods. However, The activities of $\alpha$-fucosidase, $\alpha$ -mannosidase, $\beta$-galactosidase and N-acetyl- $\beta$-D-glucosaminidase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may play a positive role in promoting of sperm function and glycosidase activity in vitro in pig.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.52-55
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• 2017

The aim of this study was to report a successful pregnancy using completely immotile frozen-thawed spermatozoa selected by laser. A single laser shot was used to detect the presence of viable immotile spermatozoa in fresh and frozen-thawed testicular spermatozoa. The viability rate was 55.8% after the laser detection, and cryopreservation was carried out immediately. The thawing test was performed on the day of oocyte pick-up, and no motile sperm were observed after extending the culture for another 4 hours, while a survival rate of 39.8% was detected using the laser. In all, five mature oocytes were injected, resulting in four cases of normal fertilization (80%) on day 1. Further, two high-quality day 3 embryos were transferred, which resulted in a singleton pregnancy. Our study demonstrates that completely immotile spermatozoa are worth cryopreserving for further intracytoplasmic sperm injection, which provides a new insight into male fertility preservation in cases of completely immotile spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1716-1721
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• 2006

The present study was conducted to observe the effect of osmolality of glycerolated TEST-yolk glycerol extenders on post-thawing sperm kinematics of ram spermatozoa of the native Malpura breed maintained in a semi-arid tropical environment. Good quality semen obtained from adult rams was pooled, split and diluted to 1,000 million spermatozoa per ml in complete TEST-yolk-glycerol extenders of 900, 1,200, 1,500 and 1,800 mOsm/kg osmolality. Diluted semen samples were loaded in 0.25 ml straws and cooled down to $-125^{\circ}C$ freezing temperature at the rate of $-25^{\circ}C$ per minute under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at $50^{\circ}C$ in a water bath for 10 seconds and sperm kinematics of the frozen-thawed spermatozoa were assessed by a computer-assisted sperm analysis technique. Osmolality of diluent had no significant effect on post-thawing % motility, % rapid, % medium and % slow moving frozen-thawed spermatozoa but significantly (p< 0.05) affected the % linearity and % straightness. The post-thawing % motility and % rapid motile spermatozoa were highest in samples extended in diluent of 1,500 mOsm/kg osmolality and lowest in 900 mOsm/kg. The curvilinear velocity of spermatozoa was significantly (p<0.05) higher for samples extended in 1,800 mOsm/kg, compared to those in 900 and 1,200 mOsm/kg, but the effect was not significantly different to those extended in diluent of 1,500 mOsm/kg osmolality. The study indicated that ram spermatozoa could tolerate a wide osmolality range for dilution in the complete TEST-yolk-glycerol extender for their cryosurvival. The highest recovery of motile spermatozoa following thawing was achieved in samples extended in the TEST-yolk-glycerol diluent of 1,500 mOsm/kg osmolality.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.121-126
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• 2007

The objective of this study was to establish the optimal conditions for hypoosmotic swelling (HOS) test to assess the functional integrity of the membranes of boar fresh or frozen/thawed spermatozoa. When pooled semen sample was incubated for 30 min at $37^{\circ}C$ with different test solution of varied osmolarity, the highest percentage of HOS positive spermatozoa was observed in a 150 mOsmol fructose/Na-citrate solution (33.6%). Incubation time did not affect significantly the score of HOS positive spermatozoa observed in a 150 mOsmol fructose/Na-citrate solution at $37^{\circ}C$, but the osmolarity affected the score of HOS positive spermatozoa under the same condition above. Fresh semen was significantly better than frozen/thawed semen in semen parameters evaluated such as motility, viability, membrane integrity and lipid peroxidation (p<005). In the relationships of sperm parameters, motility vs viability, motility vs membrane integrity and viability vs membrane integrity were positively correlated ($0.82{\sim}0.94$) but lipid peroxidation vs other estimated factors was negatively correlated ($- 0.90{\sim}- 0.98$). Among the evaluation methods, motility vs Viability, motility vs membrane integrity and lipid peroxidation vs other estimated factors were significantly correlated (p<0.05). These results of this. study indicate that the optimal condition of HOST in boar spermatozoa is a 150 mOsmol fructose/Na-citrate solution for 30 min incubation at $37^{\circ}C$ and HOST can substitute the examination of motility, viability and lipid peroxidation.