• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.251-258
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• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.195-201
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• 2003

This study was carried out to establish most suitable freezing condition, to evaluate the different condition of freezing and thawing rates on the viability and motility of frozen canine spermatozoa. The ejaculated semen was added to obtained 200∼400 ${\times}$ 10$^{6}$ /$m\ell$ with extender I and was cooled to 4$^{\circ}C$ over 30, 60 and 120 minutes. And then, semen was diluted with extender II containing 4, 6 and 8％(v/v) glycerol for 60 min, respectively and packaged in 0.5$m\ell$ straw, equilibrated far 30, 60 and 120 min at 4$^{\circ}C$ and cryopreserved in liquid nitrogen vapor at different distance(3, 5, 7 and 9 cm, respectively), plunged into nitrogen tank. Samples were thawed by placing straws into 27, 37, 47, 57$^{\circ}C$ water bath for 120, 20 and 12 sec, respectively. The results were as follows; 1. The survival and motility rate immediately post-thawing was significantly higher in samples frozen in 4％ glycerol than 6 or 8％ glycerol(P< 0.05). 2. According to equilibration time at 4$^{\circ}C$, the survival and motility rate immediately post-thawing was significantly higher in samples frozen after 60 min equilibration than 30 or 120 min equilibration(P<0.05). 3. Freezing in distance of 5 cm from liquid nitrogen yield better survival and motility rate than the others(3, 7 or 9 cm)(P<0.05). 4. The effect of thawing rate on sperm survival were higher when the thawing was done at 37$^{\circ}C$ for 120 sec(P<0.05).

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.199-206
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol ($39.85%{\pm}11.41$) than in 5% glycerol treatment ($18.08%{\pm}1.61$). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol ($34.12%{\pm}11.02$) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

• Korean Journal of Poultry Science
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• v.39 no.4
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• pp.291-295
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• 2012

The use of methylacetamide (MA) as a cryoprotective agent for freezing Korean Native Black rooster Ogye semen was examined with artificial insemination. The diluted Ogye semen with HS-1 was subjected for 2 step dilution method of cryopreservation in which the final concentration of MA was adjusted to 7.5%. The sperm viability after thawing was reduced from $95.17{\pm}0.93%$ to $55.93{\pm}1.38%$ which was confirmed by live-death analysis based on Fluorescence-Activated Cell Sorting (FACS). The rates of fertilized eggs with fresh or frozen-thawed semen were reduced from $94.98{\pm}3.93%$ to $66.36{\pm}8.43%$ at day 7 with significant difference. However, the hatching rates of experiments at day 21 did not shown difference between $92.64{\pm}2.33%$ and $90.45{\pm}8.05%$ (P<0.05). With these results, the utilization of MA for freezing of Ogye spermatozoa could affect on viability of frozen-thawed semen but not on the fertility of lain eggs and hatchability of fertilized eggs and also provide possible tools of freezing for poultry genetic resource conservation.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.3
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• pp.199-205
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• 2020

Cryopreservation of semen is useful for animal breeding via artificial insemination (AI). However, the use of frozen-thawed boar semen is limited due to cryodamage. The aim of this study was to investigate the effects of different concentrations of MitoTEMPO (a mitochondria-targeted antioxidant) in lactose-egg yolk (LEY) extenders on kinetic characteristics of frozen-thawed boar sperms. Semen samples were collected from mature Duroc boars (2~3 years old) and cryopreserved in LEY extenders containing 0, 0.5, 5, 50, and 500 μM MitoTEMPO. The kinetic characteristics of frozen-thawed sperms were determined 0 and 30 min after thawing using computer-assisted sperm analysis (CASA). Results indicated that sperm motility immediately after thawing was significantly higher with 5 and 50 μM (50.46±2.71% and 46.96±2.66%, respectively) than with 500 μM MitoTEMPO (35.40±2.95%) (P<0.05). However, there were no significant differences in other kinetic characteristics except motility. In conclusion, the addition of MitoTEMPO to the sperm freezing extender may have a beneficial effect on motility of post-thawed boar semen.