• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2001.05a
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• pp.30-30
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• 2001

• Korean Journal of Fisheries and Aquatic Sciences
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• v.41 no.4
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• pp.267-271
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• 2008

We experimentally determined the physico-chemical properties of seminal plasma as well as the sperm cryopreservation techniques of masou salmon, Oncorhynchus masou masou. Seminal plasma contained $18{\pm}1mmol/L$ potassium, $144{\pm}4mmol/L$ sodium, $116{\pm}3mmol/L$ chloride, $83.2{\pm}3.1mg/L$ calcium, $14.8{\pm}0.7mg/L$ magnesium, $45{\pm}9mg/L$ glucose, and $1.0{\pm}0.0g/L$ total protein. The osmolality and pH of seminal plasma were $287{\pm}7\;and\;7.7{\pm}0.1mmol/kg$, respectively, and the spermatocrit was $28{\pm}2$. The rate of embryonic survival at the eyed-stage and the hatching rate were highest in 10% methanol with 300 mM glucose. Compared to DMSO or glycerol, methanol served as a better cryoprotectant of masou salmon sperm.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.3
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• pp.353-358
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• 1998

Experiments were performed to find out the physico-chemical properties of milt, and morphological changes of cryopreserved spermatozoa in tiger puffer, Takifugu rubripes. The average number of sperm and spermatocrit in milt stripped were $9.81{\pm}0.34{\times}10^{10}/m{\ell}$ and $97.8{\pm}0.8$, respectively. While total lipid concentration from seminal fluid was higher than that from sperm, total protein concentration from sperm was higher than that from seminal fluid, Na and K concentrations in sperm and those in seminal fluid were similar each other, However, glucose from sperm and seminal fluid were not detectable. Spermatozoon of tiger puffer was consisted of head, middle Piece and tail. Size of head showing horseshoe shape was $0.65{\pm}0.10{\mu}m$ in diameter and $1.35{\pm}0.30{\mu}m$ in length. The head fully containing chromatin did not have acrosome. Mitochondrion in middle piece was $0.2{\mu}m$ in average diameter and flagellum showed 9+2 structure. A few of cryopreserved spermatozoa showed morphologically loose or swollen plasma membranes.

• Development and Reproduction
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• v.11 no.3
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• pp.227-233
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• 2007

The milt properties of filefish(Thamnaconus modestus) included physical properties of sperm and biochemical properties of seminal plasma. The physical properties of milt were $0.3{\pm}0.1\;mL{\cdot}fish^{-1}$ in sperm volume, $2.6{\pm}0.1{\times}10^7\;spermatozoa{\cdotg}mL^{-1}$ in sperm concentration and $73.3{\pm}6.7$ in spermatocrit. The biochemical properties of seminal plasma contained $9.8{\pm}0.9\;mmol{\cdot}L^{-1}$ potassium, $164.0{\pm}4.0\;mmol{\cdot}L^{-1}$ sodium, $151.0{\pm}1.2\;mmol{\cdot}L^{-1}$ chloride, $14.9{\pm}0.6\;mg{\cdot}dL^{-1}$ calcium, $7.2{\pm}0.1\;mg{\cdot}dL^{-1}$ magnesium, $1.0\;mg{\cdot}dL^{-1}$ glucose, $0.1\;g{\cdot}dL^{-1}$ total protein and $1.0\;mg{\cdot}dL^{-1}$ total lipid. The osmolality and pH of seminal plasma were $322.8{\pm}2.8\;mOsmol{\cdot}kg^{-1}$ and $7.7{\pm}0.1$, respectively. The spermatozoon of filefish consisted of three parts: head without acrosome, mid-piece with five mitochondria and flagellum with "9+2" pattern. The head of spermatozoon in longitudinal section was horseshoe-shaped, and $1.3{\sim}1.6\;{\mu}m$ long and $1.0{\sim}1.3\;{\mu}m$ wide.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.23-30
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• 2018

The purpose of this study was to investigate the effects of the concentration of seminal plasma in aerobic and anaerobic conditions on the total motility(TM) and the progressive motility(PM) of spermatozoa in long term preservation of cooled equine semen. We also examine the pregnancy rates after artificial insemination using fresh, cooled or frozen semen, and different durations of cooled-preserved equine semen. In the aerobic state of cooled-preserved semen, As the increase of preserved duration to 24h, 48h, 72h, and 96h, TM tended to decrease in each of different concentrations of formalin-containing experimental group, TM tended to decrease regardless of the concentrations of SP. In different concentrations of SP, TM of without seminal plasma(SP W/O) group tended to be higher than that of SP 20%, SP 33% and SP 50%, especially TM of SP W/O group was significantly higher than other groups at 96 h (p<0.05). PM was higher in the groups of SP W/O and SP 20% than in the groups of SP 33% and SP 50% from 24 h to 72 h in cooled-preservation, especially PM of SP W/O group was significantly higher than other groups at 96 h (p<0.05). In the anaerobic condition of cooled-preserved semen, the results of TM and PM at different concentrations of SP were similar to the results in the aerobic condition although there was a difference in the ratio. The pregnancy rates of fresh-cooled, cooled-preserved and frozen semen were 66.3%, 60.7% and 34.5%, respectively, and the pregnancy rate of frozen semen was the lowest. We also found that it is possible to pregnancy after artificial insemination using 72 h cooled-preserved equine semen. There was similar of the pregnancy rates in the different month from April to August.

• Journal of Veterinary Clinics
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• v.19 no.1
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• pp.80-85
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• 2002

This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.486-492
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• 2007

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

• Fisheries and Aquatic Sciences
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• v.5 no.2
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• pp.87-96
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• 2002

The physico-chemical properties of fresh milt of marbled sole, Limanda yokohamae, brown sole, Limanda herzensteini, starry flounder, Platichthys stellatus and olive flounder, Paralichthys olivaceus among flatfishes, and the fine structure of their cryopresserved spermatozoa were investigated. The highest concentration of sperm among these four species was $3.60\pm1.35\times10^{10}/mL$ in marbled sole. Osmolality and pH of seminal plasma in four flatfish species were approximately 330 mOsm/kg and 7.6-8.1, respectively. Seminal plasma compositions showed interspecific differences. The sperm heads of marbled sole, brown sole and starry flounder were ellipsoidal and that of olive flounder was round. The numbers of mitochondria of these four species were eight in marbled sole, seven in brown sole and starry flounder, and six in olive flounder. Cross-sectional view of flagellum showed typical 9+ 2 structure in all species. Most of sperms cryopreserved with a proper method had no visible ultrastructural changes after freeze-thawing, compared with the fresh sperm, but in a few cases, swelling of their heads and midpiece regions were observed.

• Korean Journal of Animal Reproduction
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• v.11 no.1
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• pp.42-62
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• 1987

본 실험은 성숙가토의 정자와 정장의 항원성을 조사하여 면역적 불임원인 규명에 필요한 기초자료를 얻고자 시도하였다. 인공질로 채취한 정액을 원심분리하여 얻은 정자와 정장을 항원으로 사용하였으며 항원성의 측정방법은 크로마토그라프에 의한 단백질 분리, SDS-PAGE, HPLC, 한천확산법, 전기영동, 수동적혈구응집방법 및 부동화시험이었다. 얻어진 결과는 다음과 같다. 1. SDS-PAGE 전기영동시 정상가토 정장에서 약 23개의 단백질이 분리되었고, 그중 분자량이 약 20,000되는 단백질부분이 정관절제 수술한 정장에서 나타나지 않았다. 2. 정상가토 정장을 HPLC를 이용한 분석에서 3개의 peaks을 볼 수 있었고, 정관절제수술한 것에서는 peak 1에 해당하는 단백질이 소실되었다. 3. 한천확산시험에서 정상정장은 이종혈청과는 4개의 침강선을 나타냈고, 전기영동에서는 7개의 침강선을 나타냈다. 4. 이종면역에서 정자 및 정장은 항체가 상승이 용이하였지만, 동종면역시는 추가면역이 필요하였으며 개체간의 역가차이를 보였다. 정자면역한 자성가토에서는 수동적혈구응집반응을 나타냈지만, 한천확산 및 전기영동반응은 보이지 않았고, 같은 처치를 받은 웅성가토에서도 역시 같은 반응양상을 나타냈다. 5. 동종 및 이종항혈청을 이용한 교차전기영동방법으로 정장의 항원적 구성요소를 구별할 수 있었다. 사출된 정액에서 분리된 정장은 일부 항원을 포함하고 있었으며 이는 성숙한 정자에서 기인한 것으로 사료되었다.

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.168-172
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• 2007

In the present study, attempts were made to find out the physico-chemical properties of milt and the sperm motilities in various osmotic conditions using Panther puffer, Takifugu pardalis. The average concentration of sperm in the milt was $12.1{\pm}3.2{\times}10^9/mL$. pH and osmolality of seminal plasma were $8.2{\pm}0.3$, $385.5{\pm}12.5mOsm/kg$, respectively. Spermatozoa were immotile when the milt was mixed with solutions (electrolyte or non-electrolyte) of lower osmolality than the average seminal plasma osmolality ($385.5{\pm}12.5mOsm/kg$), but became motile after mixing milt with hyperosmotic solutions.