• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Journal of Life Science
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• v.29 no.4
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• pp.395-401
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• 2019

Phthalate is a chemical endocrine disrupter and interfere with the action of hormones, estrogens, androgens and thyroid hormones. It also affect cardiovascular, metabolic, immune and reproductive system in the human and animals. Curcumin is antioxidant, anti-inflammatory activity and -cancer properties in the human. We studied whether phthalates damage viability, mitochondrial activity and membrane integrity of sperm in boar semen. We also treated curcumin with/without phthalates in the boar semen. Fresh boar semen was treated with phthalates and/or curcumin for examining sperm characteristics. Sperm characteristics, sperm motility, viability, mitochondrial activity, and membrane integrity were determined during storage of boar semen. Sperm motility and viability in dose-dependent manner decreased by di-n-butyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP, p<0.05). Phthalates also decreased mitochondrial activity and membrane integrity of sperm (p<0.05). However, sperm motility and viability were higher than untreated-curcumin when DBP, MBP and DEHP treated with a curcumin in boar semen (p<0.05). Mitochondrial activity and membrane integrity of sperm were higher in DBP- and MBP-treated semen with curcumin (p<0.05). In conclusion, phthalates can damage sperm viability and quality during the boar semen storage, and curcumin may protect the boar sperms from phthalates during storage term.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.4
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• pp.445-449
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• 1998

Semen quality was compared between the local Katjang and the cross-bred (local Katjang ♀ ${\times}$ German Fawn ♂) bucks. There were on significant genotypic differences in semen characteristics of concentration (first ejaculate : $6.19{\pm}1.30$ -versus $6.33{\pm}1.40{\times}10^9/ml;$second ejaculate: $5.82{\pm}1.10$ - versus $5.68{\pm}1.45{\times}10^9/ml$, for Katjang and the cross-breds, respectively), percentage live (first ejaculate: $77.61{\pm}1.33%$ versus $77.81{\pm}0.53%$; second ejaculate: $81.97{\pm}1.59%$ versus $82.74{\pm}0.96%$, for Katjang and cross-breds, respectively) and percentage of normal sperms (first ejaculate: $12.54{\pm}3.88%$ versus $26.45{\pm}3.83%$; second ejaculate: $38.68{\pm}3.65%$ versus $28.54{\pm}4.38%$, for Katjang and cross-breds, respectively), with the exception of seminal volume and sperm motility. Means of all variables were within the values reported for other goat breeds, In contrast, the differences in semen characteristics between the first and second ejaculations of both genotypes were more distinct, the second ejaculations always had more volume, more normal sperms and better sperm motility but less sperm concentrations. Removing the seminal plasma and replacing it with tris-citrate buffer greatly prolonged the viability of sperms of both genotypes when stored at $5{^{\circ}C}$. Sperm motility seens to be a good indicator of sperm viability. However, the sperms of the corss-bred bucks withstood the washing process better and their swimming abilities were superior ($8.12{\pm}0.46mm/min$) when compared to those of the local Katjang breed ($5.42{\pm}0.49mm/min$). The higher content of calcium ions in their seminal plasma (first ejaculate: $10.5{\pm}0.8$ versus $10.6{\pm}0.8mg/100ml$;second ejaculate: $15.3{\pm}0.8$ versus $16.1{\pm}0.8mg/100ml$, for Katjang and cross-breds, respectively) means that in natural matings the sperms of the cross-breds would be at an advantage compared to those of the local Katjang, since calcium ions reportedly initiate acrosomal reactions.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.207-213
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• 2017

Objective: This study investigated the prevalence of infections with human papillomavirus, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium in the semen of Korean infertile couples and their associations with sperm quality. Methods: Semen specimens were collected from 400 men who underwent a fertility evaluation. Infection with above five pathogens was assessed in each specimen. Sperm quality was compared in the pathogen-infected group and the non-infected group. Results: The infection rates of human papillomavirus, C. trachomatis, U. urealyticum, M. hominis, and M. genitalium in the study subjects were 1.57%, 0.79%, 16.80%, 4.46%, and 1.31%, respectively. The rate of morphological normality in the U. urealyticum-infected group was significantly lower than in those not infected with U. urealyticum. In a subgroup analysis of normozoospermic samples, the semen volume and the total sperm count in the pathogen-infected group were significantly lower than in the non-infected group. Conclusion: Our results suggest that infection with U. urealyticum alone and any of the five sexually transmitted infections are likely to affect sperm morphology and semen volume, respectively.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.31-39
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• 2013

Alpha-tocopherol as an antioxidant acts in preservation of chilled semen by preserving cell membrane damage from lipid peroxidation. Optimum concentrations of ${\alpha}$-tocopherol in egg yolk-citrate (EYC) extender need to be studied in crossbred bull's semen. Different concentrations of ${\alpha}$-tocopherol viz. 0, 1, 2, 4 and 6mg per ml of extender were used. Semen was collected once a week from four bulls used to regular collection, aged 4 to 7 years, weighing 320 to 450 kg, and with body condition score 4 to 4.5 and scrotal circumference 23 to 32 cm. Semen was evaluated routinely and sperm morphology was viewed under light microscope at ${\times}1,000$ magnification after fixing with buffered formal saline. Over 90% had normal head, acrosome, mid-piece and tail. Semen was diluted with egg-yolk-citrate extender to produce $15{\times}10^6$ spermatozoa/ml and 0, 1, 2, 4 and 6 mg/ml ${\alpha}$-tocopherol were added. The semens amples were kept at $8^{\circ}C$. Sperm motility and viability were examined daily up to 5 days under light microscopy at ${\times}200$ magnification. Sperm viability was acceptable (${\geq}40%$) up to the $4^{th}$ day with all concentrations of ${\alpha}$-tocopherol and up to the $5^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. Sperm motility was acceptable (${\geq}40%$) up to the $3^{rd}$ day irrespective of ${\alpha}$-tocopherol concentration, and up to the $4^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. It is suggested that the lifespan of chilled semen may be extended up to 4 days by adding 2mg/ml ${\alpha}$-tocopherol.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.4
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• pp.199-206
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• 2016

Objective: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results: The sperm DFI showed a significant correlation (r=-0.347, p< 0.001) with sperm motility and morphology (r=-0.114, p< 0.05) but not with other semen parameters. The DFI ($11.5%{\pm}2.0%$) of semen samples was significantly reduced by DGC ($8.1%{\pm}4.1%$) or MACS alone ($7.4%{\pm}3.9%$) (p< 0.05). The DFI was significantly further reduced by a combination of DGC and MACS ($4.1%{\pm}1.3%$, p< 0.05). Moreover, the combination of DGC and MACS ($1.6%{\pm}1.1%$, p< 0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC ($4.4%{\pm}3.2%$) or MACS alone ($3.4%{\pm}2.2%$). Conclusion: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

• The Korea Journal of Herbology
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• v.33 no.3
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• pp.25-35
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• 2018

Objectives : Lepidii seu Descurainiae Semen has been frequently adulterated with the seeds of several inauthentic plant species. However, the accurate identification of these plant seeds is very difficult. To develop a reliable genetic authentication tool for Lepidii seu Descurainiae Semen, we analyzed matK sequence. Methods : To obtain the matK sequences of plant materials, genomic DNA was extracted from 24 samples and PCR amplification was carried out using matK-AF/matK-8R universal primer set and matK-LDSF/matK-LDSR primer set. For identifying species-specific nucleotides and phylogenetic analysis, matK regions were sequenced and comparatively analyzed by the ClustalW and Maximum Likelihood method. Results : We developed a new primer set to amplify matK region in Lepidii seu Descurainiae Semen and closely related plant samples. From the comparative analysis of matK sequences, we identified species-specific marker nucleotides for D. sophia, L. apetalum, L. latifolium, E. cheiranthoides, E. macilentum, and D. nemorosa, respectively. Furthermore, phylogenetic analysis revealed clear classification depending on the species. These results indicated that the matK sequence obtained a new primer set in this study was useful to identify Lepidii seu Descurainiae Semen in species level. Conclusions : We developed a primer set and identified species-specific marker nucleotides enough to distinguish authentic Lepidii seu Descurainiae Semen and adulterants at the species level based on the matK sequences. These genetic tool will be useful to prevent adulteration and to standardize the quality of Lepidii seu Descurainiae Semen.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1276-1281
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• 2010

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than $3{\times}10^3$ sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

• Korean Journal of Agricultural Science
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• v.47 no.3
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.13-17
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• 2016

The purpose of this study was to examine the effects of taurine and vitamin E on sperm characteristics damaged by bromopropane (BP) in pig. We evaluated toxicity of BP on viability, membrane integrity and mitochondrial activity of spermatozoa. 1-BP (0, 2.5, 5.0, 10, and $50{\mu}M$), 2-BP (0, 2.5, 5.0, 10, and $50{\mu}M$), taurine (0, 5.0, 10, and $25{\mu}M$) and vitamin E (0, 50, 100, and $200{\mu}M$) were treated in fresh boar semen for 6 h. 10 and $50{\mu}M$ of 1-BP and 2-BP inhibited sperm viability, membrane integrity and mitochondrial activity in fresh boar semen (P<0.05). $25{\mu}M$ of taurine increased sperm viability and membrane integrity (P<0.05), $100{\mu}M$ of vitamin E enhanced viability and mitochondrial activity of sperm (P<0.05). Finally, $10{\mu}M$ of 1-BP and 2-BP was co-treated with taurine ($25{\mu}M$) and vitamin E ($100{\mu}M$) in the fresh boar semen. The co-treated samples did affected viability, membrane integrity and mitochondrial activity of sperm. In conclusion, taurine and vitamin E can improve and maintain sperm quality in fresh boar semen.