• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.486-494
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• 2006

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.

• Journal of Veterinary Clinics
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• v.21 no.4
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• pp.363-368
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• 2004

To investigate the effects of seeding during freezing procedure on post-thaw viability, motility, and acrosome integrity of boar spermatozoa, semen from 5 Yorkshire boars were collected for this experiment. Raw semen were diluted with Merck I, subsequently added with cooling diluent containing lactose and egg yolk and with freezing diluent containing glycerol. The diluted semen were frozen on the rack in the styrofoam box filled with liquid nitrogen at the distance of 5 cm or I cm above LN2 level. Seeding was performed to only a group of straws frozen at 5 cm away on the surface of LN2. The frozen semen were thawed in $50^{\circ}C$C water and the viability and local motility were analyzed by sperm analysis imaging system. A part of thawed semen was taken for the examination of morphology of apical ridge of the acrosome to compare with the effect of seeding between the seeding-treated and non treated groups. I. Post-thaw viability was considerably higher in seeding-treated sperm than non-seeding group (p<0.01), however, no difference of local motility was obtained among the groups. 2. At three hours after thawing, viability was also higher in seeding-treated group than non-treated group (p<0.05), along with no difference of motility among the groups. 3. Higher normal acrosome integrity was obtained in the seeding-treated sperm than non-treated groups (p<0.01). 4. Between non-seeded groups, higher normal acrosome integrity was obtained in the sperm group frozen at 5cm upper on the surface of LN2 than that frozen at 1cm away (p<0.01). These results indicated that seeding treatment during freezing boar spermatozoa was beneficial to post-thaw viability and normal acrosome integrity.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.337-342
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• 2018

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

• Journal of Veterinary Clinics
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• v.33 no.6
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• pp.356-361
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• 2016

During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of long-term preserved boar semen at $17^{\circ}C$. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to $20{\times}10^6sperm/ml$ in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at $17^{\circ}C$ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain $Fertilight^{(R)}$, respectively. Sperm ROS level and LPO were assessed using the 2', 7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at $17^{\circ}C$ for a period of 168 h.

• BMB Reports
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• v.33 no.6
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• pp.510-513
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• 2000

Boar sperminogen was purified from the acid extracts of the washed epididymal spermatozoa by gel filtration through a Sephadex G-100 column, followed by preparative SDS-PAGE. The 32 kDa sperminogen band was sliced out from the preparative SDS-PAGE and 32 kDa sperminogen was eluted from the gel matrix. The purified 32 kDa sperminogen was subjected to amino acid composition analysis. The amino acid composition of the 32 kDa boar sperminogen showed significant differences from that of either boar proacrosin or ${\beta}-acrosin$, which signifies that 32 kDa sperminogen might not be a breakdown product of proacrosin-acrosin system and that the 32 kDa sperminogen is a different protein from proacrosin-acrosin system.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.9-15
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• 2017

The present study was aimed to determine the effect of green tea extract (GTE) and beta-mercaptoethanol (${\beta}$-ME) supplementation in boar sperm freezing extender on sperm motility, viability and reactive oxygen species (ROS) level. Experimental groups were allocated into Lactose-egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/L GTE in LEY) and ${\beta}$-ME ($50{\mu}M$ ${\beta}$-ME in LEY). Spermatozoa extended with LEY were cooled to $5^{\circ}C$ for 3 h and then kept at $5^{\circ}C$ for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM (final sperm concentration: $1{\times}10^8/mL$). Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. Following thawing at $37^{\circ}C$ for 25 sec, sperm viability and ROS level were measured using fluorescent double stain Fertility(R) and cytometry, respectively. Motility and viability of GTE supplemented-group were higher than those of control and ${\beta}$-ME without significance. ROS level in GTE group showed significantly lower than control (P < 0.05). In conclusion, GTE supplementation in boar sperm freezing extender can reduce ROS generation during freezing.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.86-86
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• 2001

The aims of this work were to determine the acrosin activity and to evaluate the structural and functional integrity of AS boar spermatozoa. The acrosin activity of spermatozoa were 5.40, 4.10 and 3.40 mIU/10$^{6}$ sperm in raw, extended and frozen semen respectively , which differed significantly each other (P<0.05). After the raw and extended semen were exposured to cold and thermal shock, the acrosin activities of spermatozoa in the raw semen were 5.39, 5.21 and 5.29 mIU/10$^{6}$ sperm for control (non-shock), cold shock and thermal shock, and those of extended semen were 4.21, 3.98 and 4.00 mIU/10$^{6}$ sperm. This value among treatments did not differ significantly. The acrosin activities of spermatozoa in the extended and stored semen were 3.27, 3.52, 3.46 and 3.23 mIU/10/suup 6/ sperm, while hypo-osmotic test(HOST) values were 56.5%, 64.7%, 66.0% and 56.0%, following 4 days storage at 4$^{\circ}C$, 17$^{\circ}C$ , $25^{\circ}C$ and 37$^{\circ}C$, respectively. The results at 17$^{\circ}C$ and $25^{\circ}C$ appeared to be best compared with the other storage temperatures.

• Animal cells and systems
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• v.5 no.3
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• pp.199-203
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• 2001

Sperminogen was purified from the acid extracts of boar spermatozoa and partial peptide sequence of the 34-36 kd sperminogen was determined. Acid extracts of boar spermatozoa was gel-filtered through Sephadex G-75, and the 34-36 kd sperminogen was purified by preparative SDS-PAGE. The sperminogen bands were sliced out, and 34-36 kd sperminogen were eluted from the gel fragments and was subjected to peptide sequencing. Since the amino termini were blocked for Edman degradation method, internal amino acid sequences of the eluted 34-36 kd sperminogen were obtained from CNBr-digested peptides of sperminogen. Among several bands resolved on tricine SDS-PAGE, 14, 22 and 26 kd peptides were subjected to peptide sequencing. The ana1yzed amino acid sequences of the 26 and 22 kd peptides showed high homologies with that of the zona pellucida binding protein, Sp38, and the analyzed amino acid sequence of the 14 kd peptide showed neither sequence homology nor similarity with any known proteins.

• Reproductive and Developmental Biology
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• v.40 no.3
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• pp.27-31
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• 2016

The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.