• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.295-300
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• 2011

The objective of this study was to evaluate efficiency of methyl-beta-cyclodextrin (MBCD) in the sperm preservation of bull. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 1, 5, 10 and 20 mM MBCD before freezing process. Analysis of viability in frozen-thawed sperm was estimated by SYBR14/PI double stain, hypoosmotic swelling test(HOST) and acrosome damage with FITC-PNA, and mitochondria activation with Rhodamin123 by flow-cytometry. The sperm viability was significantly in 0 mM and 5 mM concentrations of MBCD than other groups (p<0.05). However, the HOST was significantly lower at 20 mM concentration of MBCD than other concentrations (p<0.05). In addition, acrosome damage and mitochondria activation rates were significantly lower at 20 mM concentration of MBCD than other groups (p<0.05). In conclusion, the viability of sperm was not significantly different among concentrations of MBCD 0, 5 and 10 mM, but MBCD 20 mM was significantly lower than other groups. In addition, as concentrations of MBCD was high, HOST, acrosome damage and mitochondria activation rates had a negative effect in bull sperm.

• Reproductive and Developmental Biology
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• v.40 no.4
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• pp.33-37
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• 2016

This study was conducted to evaluate effect of ${\alpha}$-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.45-50
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• 2012

The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition ($37^{\circ}C$ for 20 sec, 45 sec and $75^{\circ}C$ for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE (Viability : $60.3{\pm}2.4$, NAI : $58.6{\pm}2.2%$), TLE ($61.3{\pm}2.4$, $62.2{\pm}2.2%$) extender significantly(p<0.05) increased than that in LEY ($50.2{\pm}2.4$, $54.5{\pm}2.2%$) extender thawed at $75^{\circ}C$ for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE ($66.1{\pm}3.2$, $66.2{\pm}1.0%$) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE ($43.3{\pm}0.5%$) while that in LEY ($63.5{\pm}2.3%$) is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

• Clinical and Experimental Reproductive Medicine
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• v.51 no.1
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• pp.20-27
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• 2024

Objective: While sperm freezing (cryopreservation) is an effective method for preserving fertility, it can potentially harm the structure and function of sperm due to an increase in the production of reactive oxygen species. This study aimed to assess the impact of zinc oxide nanoparticles (ZnONPs) and selenium oxide nanoparticles (SeONPs) on various sperm functional parameters, including motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), acrosome membrane integrity (ACi), and malondialdehyde (MDA) levels. Methods: Semen samples were collected from 20 Albino Wistar rats. These samples were then divided into six groups: fresh, cryopreservation control, and groups supplemented with SeONPs (1, 2, 5 ㎍/mL) and ZnONPs (0.1, 1, 10 ㎍/mL). Results: Statistical analysis revealed that all concentrations of SeONPs increased total motility and progressive reduction of MDA levels compared to the cryopreservation control group (p<0.05). However, supplementation with ZnONPs did not affect these parameters (p>0.05). Conversely, supplements of 1 and 2 ㎍/mL SeONPs and 1 ㎍/mL ZnONPs contributed to the improvement of PMI and ACi (p<0.05). Yet, no significant change was observed in MMP with any concentration of SeONPs and ZnONPs compared to the cryopreservation control group (p>0.05). Conclusion: The findings suggest that optimal concentrations of SeONPs may enhance sperm parameters during the freezing process.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.147-154
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• 2012

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.185-194
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• 2018

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted ($1.5{\times}10^8/ml$) in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h. The cooled semen was then diluted ($1{\times}10^8/ml$) in LeIBP containing LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen ($LN_2$) vapors for 20 minutes and then plunged into $LN_2$. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.439-449
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• 2000

This study was to investigate the expression of transgene after co-injection of spermatozoon and EGFP gene into mature oocytes in cattle. From frozen semen, spermatozoa were treated by DTT with 0.03% Tween-20, freezing and thawing or 0.02% Triton X-100 to disrupt their plasma membranes. The sperm injected oocytes were co-cultured with bovine oviduct epithelial cells in CRlaa, and expression of EGFP in embryos were observbed under epifluorescent microscope. Two pronuclei were formed in oocytes injected with sperm treated by DTT (44.6%), DTT-Tween-20 (48.4%), DTT-freezing (44.4%) and DTT-Triton X-100 (42.9%). Cleavage and blastocyst formation rates of bovine oocytes which injected with sperm treated by DTT, DTT-Tween-20, DTT-freezing, and DTT-Triton X-100 were 49.1, 58.5, 43.9, and 48.4% and 10.2, 13.0, 6.8, and 6.5%, respectively. Although the most of embryos were showing mosaicism, embryos expressing EGFP gene were observed in all treated groups. Therefore, these results indicate that membrane disrupted sperm could interact with exogenous DNA, and that this procedure may be useful to introduce foreign gene into bovine oocytes.

• 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.