• Journal of Embryo Transfer
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• v.12 no.2
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• pp.141-149
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• 1997

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

• KSBB Journal
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• v.22 no.5
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• pp.313-317
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• 2007

To enhance the growth of cryopreserved cells of transgenic Nicotiana tabacum, Pluronic F-68 was supplemented in a recovery medium during post-thaw period. As cryoprotective agents, 1 M sucrose, 0.5 M glycerol and 0.5 M dimethyl sulfoxide (DMSO) were added before freezing steps. The post-thaw growth of the cells was improved with Pluronic F-68, ranged from 0.1 to 10 g/L. The interactions of Pluronic F-68 with the cells were confirmed by the changes of hydrophobicity or permeability of the cells. Pluronic F-68 did not show any effect on the activity of $\beta$-glucuronidase (GUS) in all treatments. Therefore, the addition of Pluronic F-68 in a recovery medium was found to be beneficial to enhance the post-thaw growth of cryopreserved transgenic tobacco cells without affecting the production of recombinant protein.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.11
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• pp.1553-1558
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• 2002

This study was carried out to obtain informations regarding the effect of N-acetyl-D-glucosamine in the LEY (lactoseegg yolk) diluent according to incubation time in 5 ml maxi-straw and the effects of freezing rate, thawing temperature and thawing time in the LEN (lactose-egg yolk and N-acetyl-D-glucosamine) diluent on acrosome morphology and motility of frozen-thawed boar sperm. The study showed that the LEN diluent was higher post-thaw NAR (normal apical ridge) acrosome than the LEY diluent for 0.5 h incubation at 37$^{\circ}C$. However, there were no differences between the LEN and LEY diluents on post-thaw sperm motility according to incubation time. The straws frozen from 5.0 cm (20$^{\circ}C$/min) to 17.0 cm (1$^{\circ}C$/min) above the liquid nitrogen surface did not show any significant differences on post-thaw sperm motility. However, the straws frozen above 5.0 cm from the liquid nitrogen surface were higher NAR acrosome than those frozen above 17.0 cm. The post-thaw percentages of motile sperm and NAR acrosome were significantly higher (p<0.05) for the maxi-straws submerged for 40 or 45 sec in a 52$^{\circ}C$ water bath than for 30, 35, 50 or 55 sec. The mean sample temperatures of maxi-straws after 40 or 45 sec submersion were 20.7 or 26.4$^{\circ}C$. In conclusion, the sample temperature of the thawed semen was very important for post-thaw sperm survival in the LEN diluent of 5 ml maxi-straw. When the temperature of the thawed semen was 20.7$^{\circ}C$, the percentages of motile sperm and NAR acrosome were highest.

• Korean Journal of Veterinary Research
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• v.34 no.4
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• pp.851-855
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• 1994

The semen from four male dogs which had been proven to be fertile in the past were frozen in a deep freezer at $-60^{\circ}C$ by simple freezing method using methanol and preserved at the same teperature for from 7 to 10 days. The semen were inseminated to 7 female dogs in estrus to find out the usability of this freezing method in artificial insemination for dogs. In addition, post-thaw motility and viability of sperm from two male dogs which had been fertile were also evaluated to investigate individual difference. Successful pregnancy was obtained by artificial insemination with canine semen frozen at $-60^{\circ}C$ by simple freezing method using methanol, namely, 3 bitches among 7 bitches which had been inseminated delivered puppies(42.8%). The average litter size of the whelping dogs were 4.3 puppies. The average post-thaw motility of canine sperm in the cases of conception was showen higher than those of non-conception(65.0% vs. 42.5%), along with the, same result in the average post-thaw viability between the two groups(53.3% vs, 27.5%). Individual difference of post-thaw motility and viability was obtained between two fertile dogs(p<0.05).

• Journal of Veterinary Clinics
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• v.21 no.4
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• pp.363-368
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• 2004

To investigate the effects of seeding during freezing procedure on post-thaw viability, motility, and acrosome integrity of boar spermatozoa, semen from 5 Yorkshire boars were collected for this experiment. Raw semen were diluted with Merck I, subsequently added with cooling diluent containing lactose and egg yolk and with freezing diluent containing glycerol. The diluted semen were frozen on the rack in the styrofoam box filled with liquid nitrogen at the distance of 5 cm or I cm above LN2 level. Seeding was performed to only a group of straws frozen at 5 cm away on the surface of LN2. The frozen semen were thawed in $50^{\circ}C$C water and the viability and local motility were analyzed by sperm analysis imaging system. A part of thawed semen was taken for the examination of morphology of apical ridge of the acrosome to compare with the effect of seeding between the seeding-treated and non treated groups. I. Post-thaw viability was considerably higher in seeding-treated sperm than non-seeding group (p<0.01), however, no difference of local motility was obtained among the groups. 2. At three hours after thawing, viability was also higher in seeding-treated group than non-treated group (p<0.05), along with no difference of motility among the groups. 3. Higher normal acrosome integrity was obtained in the seeding-treated sperm than non-treated groups (p<0.01). 4. Between non-seeded groups, higher normal acrosome integrity was obtained in the sperm group frozen at 5cm upper on the surface of LN2 than that frozen at 1cm away (p<0.01). These results indicated that seeding treatment during freezing boar spermatozoa was beneficial to post-thaw viability and normal acrosome integrity.

• 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.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.31-36
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• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.335-343
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• 2018

Objective: Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. Methods: Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. Results: Regardless of the subculture number, P19 ESCs showed better (p<0.05) doubling time and less ATP production than P4 ESCs and such difference was not influenced by fresh or cryopreservation. The difference between P4 and P19 ESC lines became decreased as the post-treatment subculture was increased and the six times subculture eliminated such difference. Similarly, transient but prominent difference in ROS production and apoptotic cell number was detected between P4 and P19 ESCs only at the 1st subculture after treatment, but no statistical differences between two ESC lines was detected in other observations. Conclusion: The results of this study suggest that post-thaw subculture of ESCs under the same environment is recommended for standardizing their cellular activity. The activity of cell proliferation ability and ATP synthesis can be used as parameters for quality control of ESCs.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.