• Korean Journal of Animal Reproduction
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• v.4 no.1
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• pp.75-82
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• 1980

This experiment was carried out to improve a simple and effective procedure of egg transfer which is considered to be the most useful technique for the improvementand proliferation of domestic animals. Several experiment procedures such as superovulation, surgical recovery of ovulated egg, in vitro capacitation of ejaculated spermatozoa, in vitro fertilization and culture of embryo were conducted. The results obtained in this experiment were summarized as follows: 1. In rabbits treated with PMS in combination with estradiol and HCG, 10 to 43 eggs were obtained in one rabbit and the average ovulation number of 20 rabbits was 21 eggs. 2. Six to 24 eggs (average 13 eggs) were recovered from the removed oviducts by the methods of flushing technique and the average recovery rate of 20 rabbits was 61.9 percent. 3. Most rabbit spermatozoa ejaculated by artificial vagina were capacitated in vitro by the culture of the spermatozoa with PBI medium at 37$^{\circ}C$ for 4 hours after removal of seminal plasma. 4. Normal fetilization following in vitro fetilization was observed in 88 (42.7%) of 206 eggs. 5. The number of eggs developed to 2-, 4-, and 8-cell stage following in vitro culture with PBI medium at 37$^{\circ}C$ was 26 (70.3%), 20 (54.1%) and 17 (45.9%) of 37 eggs used for in vitro culture.

• Journal of Aquaculture
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• v.21 no.1
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• pp.54-59
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• 2008

The present study aimed to find the best diluent and cryoprotectant for sperm cryopreservation of filefish Thamnaconus modestus. Two kinds of artificial seminal plasma(ASP1, ASP2), 0.3 M glucose and marine fish Ringer's solution(MFRS) were employed as diluent. Dimethyl sulfoxide(DMSO) and methanol as cryoprotectant were selected for sperm cryopreservation. Sperm was diluted at the ratio of 1:3 with diluents containing cryoprotectants and adjusted for final concentration at 5%, 10%, 15% and 20%. Mixed milt was frozen at liquid nitrogen vapor after equilibration for 5 min. The highest motility($40.5{\pm}2.8%$) and swimming speed($81.5{\pm}4.1{\mu}m/s$) of frozen/thawed sperm were observed in ASP1 diluent containing 10% DMSO and in ASP2 containing 15% DMSO, respectively. Results showed that cryopreservation with ASP as diluent and DMSO as cryoprotectant could be adopted for long term storage of filefish sperm.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.235-241
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• 2017

To preserve genetic materials, cryopreservation of the semen from live animals is the main technique to establish cryo-banking system which could be used for artificial insemination and embryo transfer. However, the population of Korean black goat (KBG) becomes to dwindle in number and is now faced genetic erosion by crossbreeding with non-native breeds in small KBG farms. In this study, simple freezing method was used to preserve frozen semen from KBG using spermatozoa of cauda epididymis (CE) and electro-stimulated semen (ES). The negative effects of seminal plasma on fresh sperm was confirmed using precipitation test of Triladyl egg yolk diluent and sperm viability after thawing was compared between CE and ES spermatozoa. When seminal plasma of fresh ES semen was washed with semen washing media (SWM), the rates of live sperm shown no significant difference between CE and ES spermatozoa before freezing. However, the survival rate of frozen/thawed CE sperm was higher than ES ($74.6{\pm}10.6%$ vs $53.8{\pm}5.2%$) with significant difference (p < 0.05). The results of longevity test on frozen/thawed sperm from CE showed healthier sperm than ES. Therefore, spermatozoa from CE could be used for cryo-banking system in KBG lines. The more studies are needed to increase survival rate of ES semen.

• Journal of Aquaculture
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• v.20 no.3
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• pp.173-177
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• 2007

An experiment was performed to obtain cryopreservation techniques of starry flounder (Platichthys stellatus) sperm. Milt obtained from 24 males were cryopreserved using two diluents, artificial seminal plasma (ASP) and Stein's solution (SS) with three cryoprotectants, dimethyl sulfoxide (DMSO), methanol, and glycerol concentrated from 5% to 20%. Post-thaw sperm activity (motility and/or speed) revealed the highest in 10% DMSO and 15% methanol in ASP and SS as diluent. Motility and speed of cryopreserved sperm were decreased according to increase of glycerol concentration. To conclude, DMSO was a better cryoprotectant than methanol or glycerol for cryopreservation of starry flounder sperm. Glycerol was incongruent cryoprotectant because of toxic to starry flounder sperm. Most cryopreserved spermatozoa without cryoprotectant showed the enlarged head with granulated chromatin and ruptured plasma membrane by freezing and thawing injuries compared with unfrozen normal spermatozoa.

• Animal Bioscience
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• v.34 no.8
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• pp.1253-1270
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• 2021

Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination and assisted reproductive technology. However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications.

• Fisheries and Aquatic Sciences
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• v.24 no.2
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• pp.63-77
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• 2021

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.2
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• pp.180-184
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• 2003

The intrauterine inseminator (IUI) was developed to provide the method of depositing dog semen into the uterine body instead of the vagina. The IUI consists of a vaginal endoscope, a balloon sheath, and injection catheter. When the endoscope is inserted into the vagina and the balloon expanded with air, the cervical os becomes visible so a injection catheter can be inserted through the cervix for deposition of the frozen-thawed semen. The efficacy of the IUI device was compared to intra-vaginal artificial insemination using semen that had been collected and frozen from pooled sperm-rich fraction of ejaculates collected from two Jindo dog donors. Aliquots of semen were extended with a Tris-egg yolk diluent, centrifuged, the seminal plasma removed, the pellet resuspended with the same diluent, and cooled to $5^{\circ}C$ over a 2 h period. A Tris-egg yolk-glycerol extender was added at $5^{\circ}C$; after 1 h, semen was loaded into 0.5 ml straws, and straws were frozen in LN vapor for 5 min, and immersed in LN for storage. The final sperm concentration for freezing was approximately $100{\times}10^{6}cells/ml$. The straws were thawed at $70^{\circ}C$ for precisely 6 sec, 1.5 ml Tris-egg yolk buffer at $38^{\circ}C$ added, and the 2 ml of thawed semen was used for a single insemination using the IUI device. Each bitch was inseminated at optimal insemination point, which was estimated by vaginal epithelial cells staining and progesterone concentration analysis. Use of the IUI device resulted in 21 of 26 females giving birth to 89 pups ($4.2{\pm}1.6$ pups per litter), while intra-vaginal AI resulted in 6 of 15 females whelping a total of 17 pups ($2.8{\pm}1.2$ pups per litter). We believe the IUI device is easier to use than previously described devices used for intrauterine insemination. In our experience the expansion of the balloon has a calming effect on the bitch that aids the inseminator. These results indicate that the IUI device was able to provide high fertility with 50 million frozen sperm per insemination and two inseminations.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.5
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• pp.809-815
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• 1997

A series of experiments were conducted to study effects of osmolality and $Ca^{2+}$ on sperm motility in marbled sole, Limanda yokohamae. Spermatozoa were immotile when the milt was mixed with solutions (electrolyte or non-electrolyte) of lower osmolality than the average seminal plasma osmolality (351 mOsm/kg), but became motile after mixing milt with an hyperosmotic solution. However, with the osmolality above 1,639 mOsm/kg spermatozoa ceased their movement. When the milt was suspended in the $Ca^{2+}$ free artificial seawater (ASW) containing ethylenglycoltriamine (EGTA), the percentage of motile spermatozoa decreased in proportion to the increase of EGTA. Most spermatozoa were quiescent in a medium containing 3 mM EGTA. The motility of spermatozoa prevented by 3 mM EGTA was recovered by the subsequent addition of $Ca^{2+}$ over the concentration of 0.25 mM. Effects of calmodulin antagonist, trifluoperazin (TFP) on spermatozoa were examined to elucidate the possible functions of calmodulin in sperm motility. TFP immobilized spermatozoa 5n ASW at the concentration of 0.5 M. These findings suggest that calcium is an effective external factor in the initiation process of sperm motility.

• Journal of Aquaculture
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• v.8 no.3
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• pp.149-157
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• 1995

In order to obtain the basic knowledges concerned to the semen preservation of aquacultural fishes, studies on the physical and chemical properties of semen, and sperm motility with the different osmotic pressures making by adding $Na^+,\;K^+,\; Mg^{++},\;and\;Ca^{++}$ to artificial seawater (ASW) were conducted in black seabream, Acanthopagrus schlegeli. Average semen volume per fish in one strip was 1.97ml and sperm concentration was $2.33\pm1.30\times10^{10}$ sperm/ml. Spermatocrit and pH of semen were $90.6\pm5.0\;and\;8.3\pm0.1$, respectively, Osmotic pressures of rearing seawater, seminal fluid and plasma were $939\pm24,382\pm70\;and\;342\pm77$ mOsm/l, and $Na^+,\;K^+$ and $Cl^-$ concentrations of seminal fluid were $169.5\pm4.5,\;4.9\pm2.2,\;156.0\pm2.0\;mM/l$, respectively. When semen were diluted by using $Na^+,\;K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, only $Na^+$ free ASW had no sperm motility. As raising osmotic pressure graduary by addition of 1M NaCl to the $Na^+$ free ASW, spermatozoa showed the high motilities in 457-1128 mOsm/l, but the low motilities in 1398-1736 mOsm/l. In the case of same treatments with 1M of KCl, $MgC1_2\;and\;CaC1_2$ to the $K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, spermatozoa revealed the high motilities in $904\~1434,\;818\~1175\;and\;956\~1343$ mOsm/l, respectively.