• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.85-91
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• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

• Journal of Embryo Transfer
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• v.31 no.4
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• pp.355-365
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• 2016

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation. To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and $1{\mu}g/ml$) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except $1{\mu}g/ml$ (p<0.001). Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or $0.01{\mu}g/ml$ AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and $0.1{\mu}g/ml$ AFP (p<0.05), but increased with $1{\mu}g/ml$ AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

• Journal of Korean Dental Science
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• v.8 no.2
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• pp.57-64
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• 2015

Purpose: This study was conducted to determine cell viability and differentiation capability of human periodontal ligament (PDL) cells and to elucidate the effects of cryopreservation on the activity of human third molar PDL cells by comparing PDL cells with and without cryopreservation. Materials and Methods: Human PDL fibroblasts obtained from immature third molars were cultured and divided into two groups. The experimental group was cryopreserved with a slow freezing rate of $0.5^{\circ}C/min$ from $4^{\circ}C$ to $-35^{\circ}C$ followed by plunging in liquid nitrogen at $-196^{\circ}C$ and cultured after fast thawing. The control group was cultured without cryopreservation. Cell viability, growth capacity and morphology were evaluated in both groups. Bivariate statistics were used to compare 2 groups and linear mixed model analysis was used to investigate the growth trends difference over time. Result: Cell viability and growth capacity were not significantly different between the 2 groups (P>0.05). Cultured cell of both groups showed fibroblast-like in appearance, and there were no significant differences in morphology between 2 groups. The mixed model analysis revealed no significant difference of growth capacity between 2 groups over time (${\beta}=-0.0009$; P=0.138). Conclusion: This study demonstrates that cryopreservation under control does not affect the biological properties of PDL cells, supporting the feasibility of autotransplantation of cryopreserved impacted third molars.

• Archives of Plastic Surgery
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• v.36 no.2
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• pp.135-139
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• 2009

Purpose: The use of an autogenous fat graft has become a common procedure in plastic surgery. However, questions remain concerning on the viability of fat cells and preservation method of aspirated fat. The purpose of this study was to examine the viability of fat cells stored at $-20^{\circ}C$ in the freeze for 1 year after harvest from abdominal liposuction. Methods: Eighteen adults (aged 24 to 65 years old, 16 female and 2 male) were recruited for this study. Harvested aspirated fat tissues were obtained by suction - assisted lipectomy and frozen at $-20^{\circ}C$ commercial refrigerator for one year (average 12.5 months). The viability off at cells in specimens were measured after thawing. The numbers of viable cells were measured on a fluorescence microscope after staining with fluorescein diacetate and propidium iodide. GPDH (Glycerol - 3 - phosphate dehydrogenase) activity was measured. Cell culture was done for 3 weeks. Results: There were no viable cells under the fluorescence microscope, no detectable GPDH activity, and no cultured cells. Conclusion: These findings suggest that aspirated fat after frozen storage for one year at $-20^{\circ}C$ freezer is inadequate to reuse.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.83-83
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• 2002

The purpose of this study was to investigate the warming temperature and exposed time on the post-thaw survival rate and viability of bovine blastocyst cryopreserved by GMP vitrification. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into the GMP straws and immersed into LN$_2$within 20 to 25 sec. The warming rate was increased 2 times of warming temperature for improvement of post-thaw survival rates. The frozen embryos were warmed either at 35 or 70$^{\circ}C$ for 1 or 2 sec and then diluted in sucrose solution. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM: TCM199 supplemented with 10% FCS) and TCM-199 for each 5 min, respectively, and then cultured in TCM199 for 24 h. The rate of re-expanded blastocyst was significantly different fer 35 and 70$^{\circ}C$ warming temporature (76.4 vs. 89.3%; P<0.05). The rate of re-expanded blastocyst at 70$^{\circ}C$ for 1 sec was significantly higher than that for 2 sec (91.1 vs. 70.9%; P<0.05). The number of nuclei counted were significantly different among control, 35 and 70$^{\circ}C$ (121${\pm}$8.5 vs. 104${\pm}$11.7 vs. 114${\pm}$10.3; P<0.05). These results indicated that the increasing of warming rate can provide high survival rates of bovine IVP blastocysts. Especially, the best viability of post-thaw blastocyst could be thaw at 70$^{\circ}C$ for 1 sec. The warming temperature and exposed time far warming was considered to be limiting factors to the viability of bovine IVP embryos. he purpose of this study was to investigate the warming temperature and expose.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.57-63
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• 2019

This study was conducted to find out the effect that ${\kappa}-Carrageenan$ has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% ${\kappa}-carrageenan$ ($64.26{\pm}0.49$) was significantly higher than control ($40.24{\pm}8.27$) (p < 0.05). RPMs of extender with 0.1%, 0.2% ${\kappa}-carrageenan$ ($57.64{\pm}6.34$, $56.47{\pm}1.35$) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% ${\kappa}-carrageenan$ ($61{\pm}8.03$) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of ${\kappa}-carrageenan$ of 0.1% ($0.81{\pm}0.05$), 0.2% ($0.85{\pm}0.05$) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.153-161
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• 1998

This study was conducted to examine the viability of nuclear transfer bovine embryos following embryo transfer. Donor embryos were treated with nocodazole to arrest their cell-cycle-stage at mitotic(M) phase. After releasing from nocodazole blastomeres were separated and transferred into the enucleated oocytes(BC), or cultured in medium with aphidicolin. Freshly cleaved blastomeres within 1.5h after cleavage(AC) and non-cleaved ones up to 3h after releasing from nocodazole(NC) were transferred into the enucleated oocytes. Blastocysts derived from nuclear transfer were transferred to Day 7~8 recipient cows. Some blastocysts were vitrified and thawed before embryo transfer. Developmental rates to the blastocyst stage were higher in AC(18.1%, P<0.05) than BC(8.6%) and NC(5.1%). Blastocyst development slightly enhanced with aphidicolin(1~2$\mu\textrm{g}$/ml) treatment(16.9~22.6%) compared to non treated control(11.1%). Survival rate fo vitrified nuclear transfer embryos after thawing was 75%(24/32). Twnety-three vitrified nuclear transfer embryos and 3 fresh ones were transferred to 23 recipients, 6 heads were pregnant and 1 male calf(24 kg) was born from a recipient cow recevied one vitrifiedthawed nuclear transfer embryo at 277 days after embryo transfer. This result suggests that the nuclear transfer embryos can developed to term after vitrification andembryo transfer.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.243-248
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• 1992

In order to examine the viability depending on rehydration process and membrane injury of Nocardia mediterranei upon lyophilization, We labeled $3^H$-thymidine in deoxyribonucleic acid of N. mediterrranei to obtain information on the mechanisms of injury caused by lyophilization. Suspensions of rehydrated cells were incubated with added DNase in a buffer solution. Extracellular radioactivity levels appeared to be high in the rehydrated solutions after lyophilization than freezing-thawing. Thus, the membrane systems were injured by lyophilization, but not ovenvhelmed. These considerations were confirmed by electron microscopy. In effects of rehydration, the cell membrane was seriously damaged by strong atmospheric pressure as soon as the inner ampule was opened, but this was not the case without admitting air under vacuum. N. rnediterranei cells, with no additives, were lyophilized and reconstituted without admitting air, virtually about 84% of the cells were viable.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.321-326
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• 2018

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

• Journal of Animal Science and Technology
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• v.66 no.5
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• pp.891-904
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• 2024

The primary cause of sperm quality decline during the freeze-thaw pathway is the peroxidation hazard caused by reactive oxygen species produced by the biological molecules of sperm. Ascorbic acid (Vitamin C) and lycopene are two potent antioxidants that operate to prevent oxidation processes. This study aimed to analyse the effects of ascorbic acid and lycopene on the motility, viability, abnormality and plasma membrane integrity of post-thawed Sapudi rams. Sperm samples were obtained and pooled from six sexually mature Sapudi rams, separated into ten equal proportions and diluted with Tris-egg yolk-glycerol (TEY) extender. Semen was supplemented with 0 (C0; L0), 1 (C1; L1), 2 (C2; L2), 3 (C3; L3) and 4 (C4; L4) mg/100 mL (1%-4%) diluent each of ascorbic acid and lycopene, respectively. Total sperm motility, viability, abnormalities and semen membrane plasma (%) were analysed after thawing. C3 and L3 extenders resulted in higher total motility (p < 0.05) compared to the other extenders, with all treatments higher than that of the control. The extender C3 (p < 0.05) exhibited the highest semen quality. Finally, the current findings show that C3 and L3 can increase the quality of post-thawed Sapudi ram spermatozoa.