• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.247-252
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• 2019

We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. According to the results from each freezing extender, the sperm membrane integrity (HOST: Hypoosmotic Swelling Test) analysis in TCGGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Glycerol 3%, Dimethylsulpoxide 3.5 M) is 59.8 ± 0.7, TCGSD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Sucrose 0.1 M, Dimethylsulpoxide 3.5 M) is 59.3 ± 0.5 were significantly higher (p < 0.05) among the experimental groups. And MMPs analysis result, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of active MMP-2 was the highest in sperms frozen in TCGSD and TCGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Dimethylsulpoxide 3.5 M), Meanwhile, sperms from the TCGGD and TCGED (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Ethylene glycol 3%, Dimethylsulpoxide 3.5 M) group showed lower level of active MMP-2 expression. Together, these results indicate that adding glycerol or sucrose to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of rabbit sperms. Therefore, these results suggest that TCGGD or TCGSD extender method for freezing-thawing of rabbit sperm increased the viability after thawing.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.41-46
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• 2010

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution I: 11% Lactose or Triladyl + egg yolk; solution II: solution I + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CIC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p<0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.339-344
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• 2000

Objective: To evaluate silane-coated silica particles (Sil-select) as an alternative to polyvinylpyrrolidone-coated particles (Percoll) for gradient separation of spermatozoa, for use in assisted reproduction. Methods: 20 normal semen based on WHO criteria were included in this study. Recovery of motile and morphologically normal spermatozoa after using two-layer Percoll and Sil-select gradient respectively was recorded. Motility, HOST (hypoosmotic swelling test) and the detection of malondialdehyde for LPO (lipid peroxidation) after 24 h of incubation at $37^{\circ}C$ in a 5% $CO_2$ incubator were compared. Results: Percoll (78.5%) and Sil-select (79.1%) showed a significant increase in the motility compared to ejaculate (60.9%) but no difference between Percoll and Sil-select. Normal sperm morphology significantly increased after Percoll (57.6%) and Sil-select (53.7%) compared to ejaculate (35.8%) but no difference between Percoll and Sil-select. No differences in the recovery of motile spermatozoa and motility, HOST and the production of malondialdehyde after 24 h incubation were found when comparing the use of Percoll and Sil-select. Conclusion: Sil-select seems to be an attractive alternative to Percoll for sperm separation in assisted reproduction.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.45-53
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• 2009

Objectives: To determinate the optimal culture condition to maintain lifespan in human sperm, we evaluated the effect of different temperature on sperm motility and viability up to 5 days in normal specimens. Methods: Ejaculated semen samples with normal semen parameters were gently washed in HEPES buffered Tyrod's-Albumin-Lactate-Pyruvate (HTALP) media. Each 5 ml of HTALP + 0.3% albumin with $1{\times}10^6$ sperm/ml was incubated for 5 days in $37^{\circ}C$, $22^{\circ}C$, and $4^{\circ}C$. The sperm motility and kinematics were analyzed using computer assisted sperm analysis (CASA), membrane integrity was assessed by hypoosmotic swelling test (HOST), and capacitation status was evaluated by chlorotetracycline (CTC) fluorescence pattern. Each parameter was measured on day 1, 3, and 5, respectively. Results: The motility, viability and live/uncapacitated pattern were demonstrated significantly in temperature- and time-dependent difference (p<0.05). While the sperm cultured for 1 day in each temperature was not significantly different, the sperm cell kept in $22^{\circ}C$ after 3 days were preserved sperm motility, viability, membrane integrity, and F pattern better than in other culture temperatures. Conclusions: HTALP can be used a basic medium for culture and longevity preservation, and sperm cell kept at $22^{\circ}C$ is beneficial for assisted reproductive techniques.

• 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.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.275-289
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• 2002

The objective of this study was to develop an in vitro assessment of sperm fertilizing capacity of bulls and investigate the factors influencing sperm function and characteristics of frozen-thawed bovine spermatozoa. in vitro fertilization (IVF), the evaluation of motility and normal morphology, HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, luminol and lucigenin-dependent chemiluminescence for the measurement of reactive oxygen species (ROS), the measurement of malondialdehyde formation for the analysis of lipid peroxidation (LPO), and the evaluation of DNA fragmentation using the method of 747-mediated nick end labelling (TUNEL) by flow cytometry were performed in frozen-thawed bovine spermatozoa. Correlations between the rates of fertilization, blastocyst formation after IVF and the values of respective assays were investigated. 1. IVF rate and blastocyst formation rate averaged 64.4% and 34.3% for spermatozoa from high -fertility bull group and averaged 18.5% and 6.2% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. Sperm motility and percentage acrosome reaction averaged 79.0% and 66.2% for spermatozoa from high-fertility bull group and averaged 40.7% and 22.9% for spermatozoa from low-fertility bull group, respectivitely. There were not different between two bull groups. 2. Luminol depenent chemiluminescence, LPO and DNA fragementation averaged 6.4, 2.0 nmol and 2.6% from spermatozoa from high-fertility bull group and averaged 6.5, 3.1 nmol and 7.4% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. There was no significant difference in lucigenin dependent chemiluminescence between two bull groups. 3. Fertilization rate was positively correlated with motility and the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between fertilization rate and the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. 4. Blastocyst formation rate was positively correlated with the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between blastocyst formation rate and motility, the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. In conclusion, these data suggest that ROS significantly impact semen quality. The assays of this study may provide a basis fur improving in vitro assessment of sperm fertilizing capacity.