• 한국양식학회지
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• 제17권1호
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• pp.8-11
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• 2004

Milt of the catfish was stripped into immobilizing solution containing 175 mM NaCl and 30 mM Tris at pH 7.8 and was successfully cryopreserved after a stepwise freezing procedure. After stepwise thawing, motility of spermatozoa was slightly lower than that of fresh sperm. Batches of 40-80 eggs were fertilized with cryopreserved spermatozoa, after thawing and activation in solution containing 50 mM NaCl, 20 mM Tris and HCl at pH 7.8; this resulted in 62.2% fertilization success, compared to 70.6 % with fresh sperm.

• 대한수의학회지
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• 제33권1호
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• pp.171-178
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• 1993

The present study was carried out to investigate the developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulse at output voltage of 2.0 kV/cm for $100{\mu}$ sec to induce cell fusion. The recovery rate and developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos was investigated. 1. The recovery rate of nuclear transplanted 2-cell embryos in normal morphology after freezing and thawing was significantly higher in rapid freezing(DMSO 4.5M) than in slow cooling(p<0.01). 2. When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant. In summary, these experiments have proved that rapid freezing method(DMSO 4.5M) is effective in nuclear transplanted 2-cell mouse embryos. If improved micromanipulation techniques and freezing are combined, nuclear transplantation technique will contribute to the improvement of productivity in livestock animals.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.59-64
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• 1998

This study was designed to evaluate the influence of pronuclear age on the survival and post-thawing development after cryopreservation of mouse embryos. Freezing and thawing were performed in the different pronuclear stages of mouse embryos after IVF. Embryos were obtained from $F_1$ hybrid mice and classified into 4 groups according to the pronuclear stage (6hr, 9hr, 12hr and 15hr after insemination). Pronuclear ova were slowly cooled in a biological freezer using 1.5M 1,2-propanediol and 0.1M sucrose as cryoprotectant. Thawing was done at room temperature and 1,2-propanediol was removed by multi-step dilutions. Both frozen-thawed embryos and control fresh embryos were cultured in vitro in Ham's F-10 medium supplemented with 4mg/ml BSA. In control group, the development rate after 48hr was 99.3%, and the complete hatching rate after 144hr was 61.3%. In experimental groups, the survival rate after thawing was 95.4% in 6hr, 88.7% in 9hr, 75.2% in 12hr and 62.4% in 15hr after insemination, the development rate after 48hr was 61.1, 77.0, 67.0 and 79.6%, respectively, and the complete hatching rate after 144hr was 25.7, 43.7, 42.2 and 60.0%, respectively. The survival rate in 15hr was significantly lower (p<0.05) compared with other groups. In vitro development rates after 48hr were similar in all groups, but complement hatching rate was significantly lower (p<0.05) in 6hr group. In conclusion, cryopreservation of mouse pronuclear ova with 2 distinct pronuclei (9hr and 12hr groups) showed better results after thawing compared with early (6hr group) or late pronuclear ova just prior to cleavage (15hr group).

• 한국동물생명공학회지
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• 제36권2호
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• pp.69-75
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• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• 한국동물생명공학회지
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• 제39권2호
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• pp.88-94
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• 2024

Background: Using cryovial for freezing dog spermatozoa provides a practical method to increase extended sperm volume and shorten the time required for equilibration by using a simple freezing techniques. The purpose of this study was to determine the optimal thawing condition for dog sperm cryopreservation using cryovials. Methods: For sperm freezing, cryovials with 200 × 10⁶ sperm/mL were cooled after the addition of tris egg yolk extender (TEY) at 4℃ for 20 min, then TEY with 4% glycerol was added and equilibrated for another 20 min before being aligned over LN₂ vapor for another 20 min and plunged directly into LN₂. Spermatozoa were thawed in a water bath at 37℃ for varying times (25 sec, 60 sec, 90 sec, and 120 sec) in the first experiment. In the second experiment, spermatozoa were thawed in a water bath at various temperatures and times (37℃ for 1 min, 37℃ for 1 min with gentle stirring, 24℃ for 24 min, and 75℃ for 20 sec). In these experiments, the effect of thawing conditions on motility parameters, viability (SYBR-14/PI), and acrosome integrity (PSA/FITC) of spermatozoa were investigated. Results: The post-thaw sperm motility parameters, viability, and acrosome integrity were not significantly different across the experimental groups. Conclusions: In this study, the characteristics of spermatozoa frozen using cryovials were not significantly affected by various thawing conditions.

• Asian-Australasian Journal of Animal Sciences
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• 제27권6호
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• pp.791-796
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• 2014

Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididymal sperm quality following cryopreservation and thawing. After routine castration, cauda epididymides were collected from 60 cat testes. The epididymal spermatozoa from 3 cauda epididymides were pooled as 1 replicate. Spermatozoa samples (16 replicates) were examined for spermatozoa quality and then randomly divided into 4 groups: 0 mM L-carnitine (control), 12.5 mM, 25 mM and 50 mM L-carnitine. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, plasma membrane integrity, DNA integrity and acrosome integrity were evaluated. The 25 mM L-carnitine significantly improved sperm motility compared with a control group (p<0.05), although this was not significantly different among other concentrations. In conclusion, supplementation of 25 mM L-carnitine in freezing extender improves cauda epididymal spermatozoa motility. The effects of L-carnitine on the levels of oxidative stress during freezing and thawing remains to be examined.

• 한국임상수의학회지
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• 제34권2호
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• 한국수정란이식학회지
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• 제14권2호
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• pp.131-137
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• 1999

This experiment was carried out to investigate the effects of osmolarity of thawing diluents, seminal plasma added in thawing diluents on the sperm viability and the effects of thawing temperature, the temparature of the thawing diluents on the sperm viability and acrosomal morphology of boar spermatozoa by the straw method. The result obtained were summarized as follows: 1. The sperm viablilty after thawing of the frozen semen was shown greater in the high osmolarity(392~492mOsm) than low osmolarity(300mOsm) in thawing diluent. The added levels of seminal plasma in thawing diluent did not affect the viability of frozen-thawed boar semen. 2. In terms of thawing temperature, the sperm viability was shown higher in the frozen semen thawed at 5$0^{\circ}C$ for one min. (p<0.01) than those thawed at 2$0^{\circ}C$ or 37$^{\circ}C$ for one min. The sperm viability was not significant at the diluent temparature of 2$0^{\circ}C$or 37$^{\circ}C$ after thawing: but the sperm viability was higher in thawing diluent at 2$0^{\circ}C$ than in that at 37$^{\circ}C$. However, the effects of thawing temperature and diluent solution on normal acrosomal rate were not significant. 3. Cleavage rates of oocytes fertilized with frozen semen were 46.4% and 43.3%, respectively, which were thawed at 5$0^{\circ}C$ for one min. and then diluted in mBTS medium at 2$0^{\circ}C$or 37$^{\circ}C$. To sum up, the sperm viability was shown greater at the high of thawing diluents of frozen boar semen. In terms of thawing conditions, the sperm viability was shown greater, when semen was thawed at a high temperature for a short time and then diluted at the same temperature as that in the straw.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.31-36
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• 1993

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.

• 한국수정란이식학회지
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• 제24권4호
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.