• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• Journal of Veterinary Clinics
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• v.19 no.1
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• pp.80-85
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• 2002

This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.57-71
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• 1983

1. Diluted chicken semen can be preserved at 2 to 5$^{\circ}C$ for 24 to 48 hr with resultant fertility of greater than 90% of that of fresh semen. Turkey semen can be preserved at 10 to 15$^{\circ}C$ for 6 to 24 hr and provide economical fertility. 2. Frozen chicken semen has given variable results; a 21 to 93% fertility ranges as compared to 92 to 94% expected with fresh semen. Highest fertility levels obtained with frozen turkey semen intravaginally inseminated have been 61 and 63% using DMSO and glycerol, respectively, as cryoprotectants. 3. The use of glycerol as a cryoprotectant reauires that its concentration in semen be reduced to less than 2% either by dialysis or centrifugation after thawing and before intravaginal insemination if optimal fertility is to be obtained. 4. The temperature at which cryoprotectants are added to semen and the time allowed for equilibration are important for subsequent fertility pre- and post-freezing. 5. The type of container used for packaging the semen, freeze or cooling rates, thaw rates and level of cryoprotectant all interact in affecting cell survival. 6. Plastic freeze straws as a packaging device for semen offers the following advantages: easy to handle, require minimal storage space, offer a wide range of freeze and thaw rates, and insemination can be made directly from them upon thawing. 7. Controlled slow cooling rates of 1 to 8$^{\circ}C$/min have thus far provided the best results for cooling chicken semen throught the transition phase change (liquid to solid) or critical temperature range of ＋5 to -20 or -35$^{\circ}C$. 8. Highest fertilities have been achieved with frozen chicken semen where a slow thaw rate (2。 to 5$^{\circ}C$) has been used regardless of the freeze rate. 9. To maintain a constant high level of fertility throughout a breeding season with frozen semen, a higher absolute number of spermatozoa must be inseminated (2 to 3 times as many) as compared to fresh semen since a, pp.oximately 50% are destroyed during processing and freezing. 10. The quality of semen may vary with season and age of the male. Such changes in sperm quality could be accentuated by storage effects. Thus, the correct number of spermatozoa may very well vary during the course of a breeding period. 11. As to time of insemination, it is best to avoid inseminating chicken hens within 1-2 hr after or 3-5 hr before oviposition; and turkey hens during or 7-10 hr before oviposition. 12. The physiological receptiveness of the oviduct at the time of insemination is a very important biological factor influencing fertility levels throughout the breeding season.

• Korean Journal of Animal Reproduction
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• v.5 no.2
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• pp.43-48
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• 1981

This experiment was carried out with the purpose to get some information about the properties of Simmental bull semen during summer season. The results obtained were summarized as follows: 1. Semen volume per ejaculation, sperm concentration and sperm viability were averaged 5.16ml, 6.6billion and 65%, respectively. 2. Percentage of motile sperm after dilution in skmmilk solution and trisbuffer for 5 days were 34.16% and 35.0%, respectively. 3. Viability of spermatozoa frozen in skimmilk extender and trisbuffer for 5 days were 20. 83% and 25.66%, respectively. 4. Percentage of live sperm, MRT and pH value were 71.8∼72.1%, 8.40∼8.21 minutes and 6.78, respectively. 5. Diluted semen showed strong resistance to the cold shock than that of fresh semen. 6. Rscovery of sperm motility after freezing for 24 hours was relatively weak.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.265-271
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• 2011

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until $5^{\circ}C$ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.133-138
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• 2001

The aim of this study was to identify the method on extended canine semen exposed to freezing as assessed by motility, survival and acrosomal changes following different freezing ramp rates. Five ejaculates collected by digital manipulation twice weekly from three dogs (Shih-Tzu) were added Tris-Egg Yolk (TE) buffer and divided into 4 aliquots according to formulation of our laboratory. After cooling to 4$^{\circ}C$ by ramp rate of 0.6$^{\circ}C$/min, the samples frozen by ramp rates of 1.6$^{\circ}C$/min to -$25^{\circ}C$, 3$^{\circ}C$/min to -35$^{\circ}C$, 8.9$^{\circ}C$/min to -7$0^{\circ}C$ and 19$^{\circ}C$/min to -11$0^{\circ}C$, respectively, and then stored in L$N_2$for 2days. Each sample was evaluated on their motility, survivability and acrosome integrity at different thawing temperature. The ramp rate of 3$^{\circ}C$/min to -35$^{\circ}C$/h for freezing and thawing temperature of 37$^{\circ}C$ obtained the highest results to improve survivability, motile spermatozoa and intact acrosome appearance than other onditions. In conclusion, we may suggest freezing semen for canine artificial insemination is more efficient with freezing at a ramp rate of -3$^{\circ}C$/min to -35$^{\circ}C$ and thawing with a water bath adjusted to 37$^{\circ}C$.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.245-250
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• 2011

The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment, semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was $357{\times}10^6sperms/ml$ for A and $97{\times}10^6sperms/ml$ for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5% vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0% vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.311-323
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• 1994

This study was conducted to observe seasonal and individual changes in semen characteristics and sperm freezability, and sperm penetration into zona-free hamster eggs in Korean native goats. Buck response and change in semen characteristics to electrical stimulations was evaluated for four seasons throughout 2 years and percentage of motile sperm and normal apical ridge acrosome was investigated after equilibration and thawing for 4 seasons with 5 bucks. Sperm penetration rate was evaluated for 4 bucks. 1. Probe insertion at depth of 7cm and repeated stimulation for 3 sec was more effective(P<0.05) in buck response and semen collection than those of other conditions. 2. Semen characteristics from electrojaculation was signficantly(P<0.005) higher in spring and fall for semen volume, in spring and summer for sperm concentration and in fall for sperm motility than those in other seasons, respectively. However, there were no differences in total sperm among seasons. 3. Buck response to electrical stimulation showed significant difference(P<0.05) among individuals in all 3 seasons except winter. Significant individual difference in semen volume was only in spring and summer, but there was no indivudual difference in sperm concentration and total sperm in all season. 4. Washing of semen before freezing treatment was greatly(P<0.05) beneficial to sperm motility after thawing, no matter whether ejaculates exhibit egg yolk coagulation or not. 5. Sperm motility after glycerol equilibration was significantly(P<0.05) low in summer semen and motility after thawing was greatly(P<0.05) higher in winter semen than in other seasons. Freezability of unwashed sperm was significantly difference among bucks, but a yearly freezability of washed sperm after chilling and thawing were no differences among bucks and percentage of normal apical ridge acrosome were not different among seasons and bucks. 6. There was no significant difference in sperm motility after thawing between egg yolk levels in summer, although 20% level gave more higher motility than 5% level. 7. In summer, 3.2% glycerol and 3-h equilibration gave greatest percentage(P<0.05) of sperm motility and normal apical ridge acrosome after thawing. 8. Sperm penetration rate into zona-free hamster eggs was not different between bucks and seasons. Overall, it is concluded that to obtain maximum sperm output and successive semen freezing by electrojaculation method, buck selection with good response in all season could be basically considered and that seasonal effect on sperm freezability was more greater than that of individual bucks.

• Journal of Life Science
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• v.30 no.1
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• pp.77-81
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• 2020

The aim of this study was to overcome some of the limiting factors that the maxi cryopreservation straw of 5 ml presents in processing boar semen. Cryopreservation of semen samples was conducted in 0.5 ml and 5.0 ml straws at two freezing rates: -140℃ in 8 minutes and 30 seconds (FR-1) and -140℃ in 14 minutes (FR-2). The straws were then thawed and the semen parameters were compared by Computer Assisted Sperm Analysis, and sperm morphology and acrosome status were examined by Coomassie blue staining. The effects of different thawing temperatures and durations were also compared, namely 37℃ for 115 sec, 50℃ for 45 sec, or 70℃ for 25 sec. In general, the FR-1 group showed higher (p<0.05) sperm viability and motility than the FR-2 group in the 5.0 ml straws. Compared to other ranges, thawing at 50℃ for 45 sec showed the highest sperm viability and motility (68.4±3.6% and 69.5±2.2%, p<0.05), suggesting that thawing temperature should be adjusted concurrently with freezing rate. Sperm morphology and acrosome integrity did not significantly differ among the groups (p>0.05). The data obtained in this study suggest that improving the freezing-thawing protocol for one artificial insemination dose straws (5.0 ml) retains the sperm's parameters from 0.5 ml cryopreservation, and is more convenient to handle, which could result in enhanced reproductive performance.