• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.190-197
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• 2020

The effects of the number of frozen-thawed ram sperm per single and double intra-cervical artificial insemination (AI) on fertility in ewes were studied. A total of 89 non-pregnant ewes were synchronized for oestrus with two doses of 100 ㎍ PGF_2α (Cloprostenol) 9 days apart. The ewes were randomly assigned to one of four groups; D200 (n = 23; double AI with 200 × 10⁶ sperm), S200 (n = 24; single AI with 200 × 10⁶ sperm), D100 (n = 24; double AI with 100 × 10⁶ sperm) and S100 (n = 18; single AI with 100 × 10⁶ sperm). Ewes were inseminated within 12 to 18 h for single AI and, within 10 to 12 h and 16 to 18 h for double AI after the onset of oestrus. The onset of oestrus ranged from 28 to 76 h (54.33 ± 1.28 h). The high percentage (29.2%) of ewes showed oestrus between 51 to 60 h. The non-return rates were highest in group D200 (56.5%) and differed significantly (p < 0.05) from group S100 (11.1%). No ewes were pregnant in group S100, and the pregnancy rates among the remaining groups did not differ. The mean gestation period was 152.8 ± 0.5 days and no difference was observed among the groups. The lambing and multiple birth rates were 100% in group D200. The single and twin lambing was highest in group D100 (33.3%) and group D200 (83.3%), respectively. Only one triplet lambing and the highest lambing size (2.2 ± 0.2) was recorded in group D200. In conclusion, double AI with 200 × 10⁶ sperm showed comparatively most practical for achieving high pregnancy rates and lambing performances in Bangladeshi ewes under field conditions.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.405-410
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• 1998

Boar semen extender Kp (Hankook Life-Science, Korea) was newly formulated by authors. This study was carried out to investigate the optimal concentrations of EDTA, Tris and citrate buffers in the Kp extender (Basic Kp) on the pH change during storage. And then the motility of boar sperm with the Kp pH stabilized (Modified Kp) was compared with those of commercial products imported into Korea such as BTS (Mini-tube. Germany; BTSg), BTS (Tri-bio, USA; BTSa) and Modena (SGI, USA). The pH values of all extenders were increased gradually with the storage days. Especially, the initial pH of Basic Kp was higher than that of BTSg, BTSa and Modena, and also higher than physiological pH of boar sperm (6.8∼7.5). When Basic Kp was added with various concentrations of EDTA (0, 0.63, 1.25 ＆ 2.37g/L), Tris (0, 0.18, 0.35, 0.71 ＆ 1.42g/L) and Citrate (0, 0.75, 0.81, 1.00, 1.25 ＆ 1.50g/L) buffers for pH down-regulation and stabilization of pH, the group added with 1.25g EDTA, 1.42g Tris and 1.00g Citrate well maintained the neutral range of pH during storage (6.88 at day1 to 7.33 at day6 in Modified Kp), Especially, the concentrations of the buffers added in Modified Kp were lower, until 1/2∼1/4 ranges, than those in Modena and other extenders. The motility of frozen-thawed boar sperm diluted with Modified Kp was significantly higher than that of Basic Kp, BTSg, BTSa and Modena (87.0% vs. 55.0∼71.0% at day1; 13.3% vs. 0∼6.3% at day6), Conclusively, Modified Kp in this experiment was kept the favorable physiological conditions in spite of low concentrations of the buffers and motility of frozen-thawed boar sperm was obtained better than that of Basic Kp and other commercial products such as BTSg, BTSa and Modena.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.95-102
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• 2000

Techniques for manipulation of spermatozoa and oocytes have been widely used for in vitro production(IVP) of Hanwoo. This study was conducted to examine the effects of theophylline and heparin on frozen-thawed Hanwoo sperm for enhancing the efficiency of IVP technique. Oocytes were inseminated with forzen bull semen treated with either theophylline or heparin for examining the effect of each substance on fertilization and subsequent development. More (P<0.05) oocytes formed pronucleus and develop to the morula and blastocyst stages after inseminated with sperm treated with heparin than after inseminated with sperm treated with theophylline. The pregnancy rate after embryo transfer was higher after heparin treatment than after theophylline treatment, but did not differ significantly. There was no significant difference of offspring delivery between two groups. In conculsion, theophylline and heparin can be used for enhancing the efficiency of IVP system for Hanwoo. Considering characteristics of these substance, theophylline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.147-151
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• 2019

Objective: The aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST). Methods: Frozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern ("a"-"g") were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed. Results: The HOST examinations showed that > 93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type "d", 98.67% in type "g", and 98.17% in type "f" spermatozoa. Conclusion: We found that the type "d" spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.77-83
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• 2006

본 연구는 개 동결 정액 융해 시 straw 크기 및 융해 속도가 융해 정자의 질(quality)에 미치는 영향을 조사하고 최적의 융해 조건을 조사하는데 그 목적이 있다. 정상적인 번식능을 가진 비글 수컷 5마리에서 정액을 채취하여 원심 분리하여 정장을 버리고 남은 정자에 동결보호제인 glycerol이 첨가된 tris-glucose-egg yolk extender를 첨가하여 동결하고 액체질소에 보관한 후 융해하였다. 동결 융해 조건에 따른 효과를 알아보기 위해 straw는 0.25 ml과 0.5 ml크기를 사용하였고 융해 조건은 $75^{\circ}C$에 10초, $55^{\circ}C$에 12초 및 $37^{\circ}C$에서 120초로 하여 융해 후 정자의 활력도(vigor), 운동성(motility), Hypo-osmotic test(HOS test)를 이용한 생존성(viability) 및 $SperMac^{\circledR}$ 염색을 하여 정자의 membrane integrity를 비교 조사하였다. 조사 결과 0.5 ml 크기의 straw를 사용한 경우 $37^{\circ}C$ 융해가 $55^{\circ}C,\;75^{\circ}C$ 융해보다, 0.25 ml 크기의 straw를 사용한 경우에는 $37^{\circ}C,\;55^{\circ}C$ 융해가 $75^{\circ}C$ 융해보다 유의적으로 높은 활력 지수 및 생존성을 보였다(P<0.05). Straw크기에 따라 비교하였을 경우 0.5 ml 군에서 유의적으로 높은 활력도, 생존성 및 membrane integrity를 보였다(P<0.05). 결론적으로 개 정액이 동결 및 융해 시 0.5ml straw를 이용하여 동결한 후 $37^{\circ}C$에서 120초 동안 융해하는 것이 최적의 조건임이 사료된다.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.1-6
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• 2012

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability ($84{\pm}1.15%$, $p$<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability ($79{\pm}3%$, $p$<0.05) than those of the X and Y sperm ($44.7{\pm}1.67$ and $41.7{\pm}1.2%$) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities ($24.3{\pm}1.2$ and $25.7{\pm}0.9%$, $p$<0.05) compared to that of the sorted Y sperm ($13.7{\pm}1.2%$) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher ($55.0{\pm}1.7$ and $45.0{\pm}1.5%$) than those of the sorted Y-sperm ($32.3{\pm}0.9%$, $p$<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.95-101
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• 2012

The objective of this study was to examine the effect of amides as a cryoprotectant for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing 5% dimethyl acetamide (DMA), 5% dimethyl formamide (DMF), 5% methyl formamide (MF) or 7% glycerol. Post-thawed sperm were evaluated for sperm motility, viability, acrosome integrity and membrane integrity. Post-thawed sperm motility was significantly higher (p<0.05) in glycerol and DMF ($64.00%{\pm}9.62$ and $59.00%{\pm}5.48$, respectively) than DMA and MF ($50.00%{\pm}3.24$ and $44.00%{\pm}4.18$, respectively). Sperm viability wassignificantly higher (p<0.05) in glycerol and DMF ($58.25%{\pm}7.35$ and $53.05%{\pm}3.77$, respectively) than others. However, for sperm motility and viability, there were no differences among glycerol and DMF. Also, swelling sperm ratio by hypo-osmetic selling test (HOST) was significantly increased (p<0.05) in glycerol and DMF treatments ($45.12%{\pm}25.08$ and $44.95%{\pm}8.58$, respectively). The percentage of capacitated sperm assessed by CTC staining, F pattern was lower (p<0.05) in DMF than others. B pattern was increased (p<0.05) in DMA, DMF and MF when compared with glycerol. AR pattern ratio was decreased (p<0.05) in glycerol and DMF when compared with DMA and MF. These results suggested that amides performed better and could be used as a cryoprotectant for semen freezing of Korean Jeju Black Bull.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.377-383
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• 2011

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into $LN_2$. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol ($72.5{\pm}5.00%$, $54.88{\pm}0.66%$ and $46.00{\pm}2.40%$; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol ($34.69{\pm}4.64%$ vs $46.00{\pm}2.40%$; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: $55.81{\pm}2.94$, $55.19{\pm}3.34$ vs $47.94{\pm}3.48%$; p<0.05 and membrane integrity: $44.94{\pm}3.51$, $46.06{\pm}2.25$ vs $40.38{\pm}1.03%$; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycerol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).