• Animal Bioscience
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• v.34 no.2
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• pp.192-197
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• 2021

Objective: The present study evaluated the preservation of ram semen at 0℃ using soybean lecithin with a Tris-fructose extender. Methods: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0℃ in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination. Results: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05). Conclusion: These results suggest that ram sperm is capable of fertilization after preservation at 0℃ with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

• Journal of Animal Science and Technology
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• v.63 no.1
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• pp.46-57
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• 2021

Fruits with antioxidant enrichment can be an economically affordable supplement for mitigating oxidative damage prone spermatozoa membrane pathologies. Computer-assisted sperm analyzer and oxidative status were utilized to evaluate the impact of watermelon (Citrullus lanatus) fortification of dextrose saline as diluent for rooster semen and fertility response of hens inseminated. Watermelon juice and dextrose saline were used to formulate diluent of 7 treatments consisting of unextended semen (positive control), 10%, 20%, 30%, 40%, 50% and only dextrose saline (negative control) designated as Treatments 1-7. Pooled semen was obtained from fertile roosters and equilibrated with diluents at ratio 1:2 in the various treatments and were evaluated using computer software coupled microscope and seminal oxidative status assay. 168 laying hens randomly divided into 7 treatment of 8 replicates and 3 hen per replicate. Hen were everted, and semen (2 × 108 Spermatozoa) deposited intra-vagina and eggs collected over 8 weeks to assess fertility and hatchability of eggs laid. The result obtained revealed that watermelon-dextrose saline rooster semen diluent enhanced progressive motility, sperm kinetics and lowered non-progressive motility in T2-T6 compared to T7 over the 3 hours of evaluation. Watermelon addition to rooster semen diluent enhance the antioxidant capacity of rooster semen and lowered lipid peroxide generation. The percentage fertility was highest in T3 (81.01%) and T4 (81.24%) with lowest value obtained in T7 (73.46%). The hatchability of eggs set of hens inseminated with undiluted semen (71.46%) was lower than values for hens inseminated with watermelon inclusive extended semen (75.71%-80.39%). The optimal inclusion of 30%-40% watermelon in dextrose saline diluent enhance rooster semen kinetics, seminal oxidative stability and egg fertility.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.207-212
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• 2009

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of $5{\times}10^5/ml$ by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen ($80{\pm}14%\;and\;43{\pm}11%$). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above $LN_2$ ($50{\pm}14%$ and 70.7% vs, 33.18% and $65{\pm}7%$ vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.181-186
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• 2011

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.127-131
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• 2001

The Jeju horse has been raised for centuries in Jeju island. Recently, as the number of this indigenous horse has been dropped dramatically, this breed became Natural Monument #347 to conserve and multiply this endangered breed. To provide the basic information for AI, sexual activity and semen characteristics in Jeju horse were investigated. Jeju horse semen was collected using Missouri style artificial vagina from fertile stallion.\\`she number of mount per ejaculation was 2..3$\pm$1.8, and the ejaculation time was 27.0$\pm$12.5 seconds. The total volume and gel-free volume of semen was 47.8$\pm$26.7 ml and 42.7$\pm$27.4ml, respectively, and the concentration of sperm and the total number of spermatozoa per ejaculation was 200.7$\pm$112.9$\times$10$^{6}$ ml and 7.6$\pm$3.9$\times$10$^{9}$ ml, respectively. The percentage of motile sperm and the number of live spermatozoa per ejaculation was 75.0$\pm$18.2% and 6.1$\pm$3.4$\times$10$^{9}$ ml, respectively, and the pH of gel-free semen was 7.3$\pm$0.2. The total percentage of abnormal sperm was 31.5%, and the percentage of sperm with abnormal head, midpiece and tail was 9.5$\pm$11.7%, 7.0$\pm$4.0% and 15.0$\pm$15.0%, respectively.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.69-75
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• 2006

The objective of the study was to assess the general characteristics and motility characteristics with Computer Assisted Sperm Analyzer (CASA) system in Jeju horse semen. Semen was collected from 5 fertile Jeju horse by use of a Missouri type artificial vagina. Semen volume and pH were recorded, and sperm concentration was determined with a hematocytometer and motional characteristics of sperm were analysed by CASA. The viability and morphological abnormalities were assessed by a vital staining. The average volume of ejaculates was 42.5 ml and the average of sperm concentration was $198.5x10^6/m1$. The motional characteristics in Jeju horse semen was showed $70.4{\pm}28.7{\mu}m/s\;for\;VAP,\;69.6{\pm}28.9{\mu}m/s\;for\;VSL,\;94.1{\pm}30.0{\mu}m/s\;fo\;VCL,\;2.3{\pm}0.7{\mu}m/s\;for\;ALH,\;7.6{\pm}1.1Hz\;for\;BCF,\;99.1{\pm}1.2%\;for\;STR,\;and\;77.1{\pm}12.7%\;for\;LIN$. The percentage of sperm with abnormal head, midpiece and tail was 4.2%, 20.6%, 4.6% respectively.

• Journal of the korean academy of Pediatric Dentistry
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• v.27 no.2
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• pp.361-369
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• 2000

Enterococcus is a normal inhabitant of the human oral cavity, the vagina, and the gastrointestinal tract. Four isolates of Enterococcus in this study were identified as E. durans. These bacteria were characterized and the interaction of these bacteria with the important oral bacteria as like S. mutans and S. oralis was studied as follows. 1. The carbohydrate fermentation test and biochemical test showed similar results in 4 isolates. 2. The susceptibility test against erythromycin, penicillin, tobramycin, ampicillin, teicoplanin, ciprofloxacin, vancomycin, gentamicin, kanamycin, and streptomycin showed to be susceptible in all four isolates. 3. The optical density of absorbance at 550 nm was 1.405 in the culture of S. mutans in disposable cuvette, whereas being 0.855, 0.867, 0.797, and 1.083 in the combined culture of S. mutans and each E. durans. 4. The mean weight of produced artificial plaque on the wires in the beaker was $1566{\pm}103mg$ in culture of S. mutans only, whereas being reduced to $44{\pm}5mg,\;41{\pm}12mg,\;34{\pm}7mg,\;and\;38{\pm}12mg$ in the combined culture of S. mutans and each E. durans. The viable cells were $2.0\times10^9$ per ml in the culture of S. mutans wheras being $2.0\times10^7\;to\;6.0\times10^7$ per ml in the combined culture S. mutans and E. durans. 5. The viable cells were $2.1\times10^8$ per ml in the culture of S. oralis, wheras being $1.4\times10^7\;to\;7.0\times10^7$ per ml in the combined culture of S. oralis and E. durans. 6. Plasmid of about 60 kb was isolated in three isolates of E. durans. These results suggested that E. durans isolated from the oral cavity inhibited the replication of S. mutans and formation of artificial plaque, while inhibiting the replication of S. oralis a little.