• Journal of Embryo Transfer
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• v.27 no.2
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.106-115
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• 2021

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.1
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• pp.39-44
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• 2014

This study aimed to find an optimal diluent and cryoprotective agent (CPA) during cryopreservation of common Korean bitterling Acheilognathus signifier sperm. The bitterling is an endangered species in Korea, and this study will enable conservation and further technical development of artificial seed production. We tested the effects of cryopreservation and toxicity by the type of diluent and CPA. The optimal combination of diluent and CPA for cryopreservation was 300 mM glucose+10% methanol, resulting in a survival rate and sperm activity index (SAI) of $96.3{\pm}1.5%$, and $3.0{\pm}0.0$ respectively, and no significant difference compared to fresh sperm. The survival rate and SAI of post-thawed sperm was $9.7{\pm}1.5%$, $0.8{\pm}0.3$ respectively, which was significantly higher than had been achieved with other diluents and CPAs.

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.173-177
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• 2007

A series of experiments were conducted to compare the effects of various diluents and cryoprotectants on the motility and survival rate in cryopreservation of the red seabream, Pagrus major sperm. Sperm was efficiently cryopreserved using 300 mM glucose as a diluent. Two cryoprotectant, dimethyl sulfoxide (DMSO) and glycerol, were added to 300 mM glucose to formulate the extenders at concentrations between 5% and 30% by volume for freezing. The highest post-thawed sperm motility and survival rate were obtained with 10% DMSO.

• Development and Reproduction
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• v.16 no.2
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• pp.77-85
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• 2012

Various cryoprotective agents (CPA) were tested to establish the best conditions for the cryopreservation of sperm from black porgy Acanthopagrus schlegeli acclimated and raised in freshwater (BFW). Survival rates of frozen/thawed sperm from BFW were higher in the order of dimethy sulfoxide (DMSO), glycerol, ethylene glycol (EG) and methanol. Sperm motility was higher in the order of glycerol, DMSO, EG and methanol. These effects were the same in thawed sperm from black porgy raised in seawater (BSW). Thus, optimum CPA for sperm cryopreservation of BFW and BSW were DMSO and glycerol where the highest survival rates and sperm motility were found at the concentration of 10%. In particular, the survival rates and motility of thawed sperm from BFW and BSW after cryopreservation using 10% DMSO were better than when cryopreserved using 10% glycerol. On the other hand, for the thawed sperm from both BFW and BSW, the longer the preservation period was, the lower the survival rates and sperm motility were. Notably, the higher the concentration of CPA was, the lower the survival rates and sperm motility were.

• Journal of Aquaculture
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• v.12 no.1
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• pp.57-62
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• 1999

Experiments were performed to study the activity and fertility of grey mullet (Mugil cephalus) sperm after the courses of cold storage and cryopreservation. The head of spermatozoon showing spherical shape was sized $1.26{\pm}0.08 \{mu}textrm{m}$ in diameter and its nucleus contained numerous granular chromatins. Flagellum of tail showed typical 9＋2 structure. Preservation of grey mullet sperm was the most effective when it was stored with serum of the same species at $0^{\circ}C$ and sperm activity index was similar in egg-tris, 0.1 M, 0.3 M and 0.5 M glucose. When grey mullet sperm were cryopreserved in MFRS as diluent with 10% dimethyl sulfoxide was effective compared with other diluents. Some of post-thawed spermatozoa showed the enlarged head and ruptured plasma membrane compared with unfrozen spermatozoa.

• Interdisciplinary Bio Central
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• v.2 no.4
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• pp.16.1-16.4
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• 2010

Cryopreservation of mouse embryos and spermatozoa has become the foremost technique for preserving large numbers of different strains of mice with induced mutations. In 1998, our mouse bank was established in the Center for Animal Resources and Development (CARD), Institute of Resource Development and Analysis, Kumamoto University, Japan, based on the Preservation, supply and development of genetically engineered animals report published by the Ministry of Education, Culture, Sports, Science and Technology. We cryopreserve mouse embryos and sperm, supply these resources, organize training courses to educate people and form part of a domestic and international network of both mutagenesis and resource centers. We currently have over 1,500 mouse strains, 842,000 frozen embryos and 26,000 straws containing frozen sperm. Moreover, we disclose information about 1,300 deposited strains. Furthermore, over 400 strains of frozen embryos or mice produced from frozen embryos and sperm are being supplied to the requesters both domestically and internationally. Additionally we hold training courses on the cryopreservation of mouse germplasm 2~3 times a year, both domestically and internationally. In the course, we teach basic reproductive engineering techniques to trainees on a man-to-man basis. We have already held 28 training courses on the cryopreservation of mouse germplasm at our center and at other institutes.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Journal of Marine Life Science
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• v.8 no.2
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• pp.115-120
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• 2023

This study aims to find out a suitable extender and cryoprotective agent (CPA) for cryopreservation and its optimum concentration in order to conduct planned artificial seed production of Korean bullhead Pseudobagrus fulvidraco and to preserve superior sperm. Experiments were designed to investigate the effects of the different combinations of three extenders (I: 300 mM glycose, II: Kurokura extender, III: Li extender), four cryoprotectants (dimethyl sulfoxide, ethylene glycol, methanol and glycerol) and four concentrations (5, 10, 15, 20%) on the cryopreservation of Korean bullhead sperm. Postthawed sperm survival rate and sperm activity index (SAI) were detected to evaluate the effects of sperm cryopreservation. The optimal combination of extender and CPA for cryopreservation of Korean bullhead sperm was extender III + 10 and 15% methanol, resulting in a survival rate and SAI of 66.9 ± 8.7, 67.3 ± 13.1% and 2.6 ± 0.4, 2.6 ± 0.5 respectively, which was higher than had been achieved with other extenders and CPAs.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.74-74
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• 2003

In present study, attempts were made to preserve abalone (Haliotis discus hannai) sperm in liquid form at low temperature, to evaluate the effect of various diluents in short-term storage on sperm, and cryopreservation procedures were optimized for the cryoprotectants as well as freezing rates, in terms of the motility and survival rate, and the ultrastructural changes of sperm after short-term storage and cryopreservation were observed. The abalone sperm reached maximum motility until about 4min after activation. The motility was constant for about 16min, after which it dropped gradually, and about 50min later all motility ceased. Threshold activation of sperm was found in 40% artificial seawater (ASW), and motility increased as the concentration of ASW increased. In Hanks balanced salt solution without calcium (Ca-Free HBSS, 300 and 400 mOsmol/kg) and 10%, 20%, and 30% ASW the sperm was immotile, and motility once again restored incompletely only in HBSS of 300 and 400 mOsmol/kg, 20% and 30% ASW after 100% ASW was added. Sperm motility was extended following 20 days of cold storage only in 70% and 100% ASW. A high motility index of 3.5-4.5 was observed for the first 8 days in 70% and 80% ASW. In other diluents sperm motility was constant less than 10 days, and the motility index was obviously lower than that of sperm in 70% and 100% ASW. After 20 days of cold storage survival rates of 10.2%-20.7% were obtained in ASW and 300 mOsmol/kg HBSS, and that in 400 HBSS (65.3%) was significantly higher than others. The constant period of sperm motility stored in 70% ASW was longer obviously than that in 100% ASW after 6 days of storage, and the time to maximum motility of sperm stored in 70% increased gradually, while the difference in which of sperm in 100% ASW was not significant. The sperm plunged into liquid nitrogen all died except that sperm using 15% glycerol as cryoprotectant restored 10.4% of motility. The highest motility index (3.4) was obtained with 5% glycerol and freezing procedure: $50^{\circ}C$/min from $20^{\circ}C$ to $-80^{\circ}C$.