• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.187-192
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• 1997

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.29-35
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• 2012

The purpose of this study was to examine the effect of growing stages of the Korean Native Striped Bull (KNSB) on the freezability and fertility of frozen-thawed semen. First, we investigated the total motility (TM) and progressive motility (PM) according to the diluent used for semen freezing. Second, we examined the effect of the age of KNSB on semen volume, TM and PM of fresh and frozen-thawed semen. Third, we examined the effect of frozen semen from the different age of KNSB on the $in-vitro$ fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl$^{(R)}$ and Tris-egg yolk extender (EYE). Semen was collected from 5 KNSB in the growing stage (15 months) and 5 adult KNSB (36 months). When Triladyl or Tris-EYE extender was used for semen freezing, there was no difference of the mean TM and the mean PM. However, the mean TM was significantly higher in Bull No. 1885 than Bull No. 4283 ($p$ <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ($p$ <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ($2.1{\times}10^9$ spermatozoa/ml) and 36-month-old ($1.8{\times}10^9$ spermatozoa/ml) bulls. For the 15-month-old and 36-month-old bulls, the mean TM of fresh semen were 93.7% and 88.3%, respectively, and the mean PM were 97.0% and 88.3%, respectively; the 15-month-old bulls showed a particularly high PM ($p$ <0.05). For the 15-month-old and 36-month-old bulls, the mean TM (56.0% and 58.0%, respectively) and the mean PM (64.0% and 70.7%, respectively) of frozen-thawed semen did not differ. The development rates of embryos after $in-vitro$ fertilization and the pregnancy rate after artificial insemination using frozen-thawed semen did not differ according to the bull's age. In summary, semen volume differed according to the bull's age, but semen concentration and survival rate, the $in-vitro$ fertilization rate, and the pregnancy rate did not differ according to the stripe bull's age. Accordingly, semen from bulls in the growing stage can be collected and frozen for the preservation and multiplication of rare livestock.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.53-60
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• 2001

This study was conducted to evaluate whether \"Royal jelly\" (RJ) added to Tris-buffer dilute contributed to supporting post-thaw viability and longevity of frozen canine spermatozoa. Two Japanese spitzs (2 to 4 years of age) were used as a semen donor. Semen was collected by manual masturbation and separated into 3 fractions. Only the sperm-rich fraction having sperm motility of more than 70%, containing sperm concentration of 2~4$\times$10$^{8}$ cells/ml and having dead or abnormal spermatozoa of less than 15% was used for the experiment. Each ejaculated semen was centrifuged at 400 $\times$ g for 5 min and then diluted in a Tris-buffer supplemented with 20 ml egg yolk (Ext I), 4% glycero1 and 1% Equex STM Paste (Ext II) or g1ycero1, Equex STM paste and RJ of various concentrations (Ext II-RJ). After freezing and thawing, viability of spermatozoa in Ext II -RJ containing 1% RJ immediately after thawing (67.5$\pm$9.6) was significantly lower than that of Ext II , Ext II -RJ containing 0.01 or 0.1% RJ (77.5$\pm$12.5, 78.7$\pm$8.2 and 80.0$\pm$6.3). However, Ext II-RJ containing 0.1% RJ yielded higher viability than Ext II, Ext II-RJ containing 0.01% at or 1% 1 h after thawing (69.5$\pm$8.1 vs. 55.0$\pm$12.9, 57.5$\pm$9.6 and 41.5$\pm$12.6; P<0.05). At 1 h after thawing, the viability of spermatozoa thawed in 7$0^{\circ}C$ (68.8$\pm$12.5) was significantly higher than that of spermatozoa thawed in 38$^{\circ}C$ (48.8$\pm$16.3), although there was no difference in the viability between both groups immediately after thawing (77.5$\pm$9.6 and 81.3$\pm$8.1). Post-thaw viability and longevity of post-thaw spermatozoa in Ext II-RJ containing 0.1% RJ was higher in those in Ext II at 1 h (65.0$\pm$12.9 vs. 42.5$\pm$12.6), 2 h (52.5$\pm$12.6 vs. 27.5$\pm$17.1) and 3 h (40.0$\pm$14.1 vs. 20.0$\pm$12.1) after thawing. These results indicated that addition of 0.1% af to Tris-buffer enhanced post-thaw viability and longevity of canine spermatozoa and this additive can be used for increasing the possibility of collision between spermatozoa and ova during insemination.emination.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.161-166
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• 2008

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were $104\times10^6/ml$ and $86\times10^6/ml$ respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at $5^{\circ}C$ was significantly lower than that at 0 hr ($30.0\pm4.1%\;vs.\;78.0\pm2.5%,\;p<0.05$), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• 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.

• Korean Journal of Veterinary Research
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• v.31 no.1
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• pp.123-130
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• 1991

The present study was performed to investigate the effect of Ca ion concentration on sperm viability and acrosome reaction rate and to evaluate the effect of treatments using caffeine and Ca-ionophore A23187 on acrosome reaction rate in frozen-thawed bull spermatozoa. Viabilities of in vitro capacitated bull spermatozoa at 0, 2.25 and 4.5 mM Ca ion concenrations were 21.00, 26.00 and 22.59%, respectively and significantly higher in Ca ion 2.25mM added group than Ca ion free group (p<0.05) and acrosome reaction rates of in vitro capacitated bull spermatozoa were 17.09, 52.15 and 47.92%, respectively and significantly high in Ca ion added groups(p<0.05). Viabilities in vitro capacitation by caffeine and Ca-ionophore A23187 in control, caffeine treated group, Ca-ionophore A23187 treated group and caffeine+Ca-ionophore A23187 treated group were 37.91, 27.67, 22.33 and 25.59%, respectively and significantly higher in control than treated groups(p<0.05), there were no significant differences among the treated groups, and acrosome reaction rates were 10.33, 37.92, 48.09 and 57.17%, respectively and there were significant differences among the groups(p<0.05), especially higher in caffeine+Ca-ionophore A23187 treated group than others.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.130-135
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• 2022

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO₂, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN₂ vapor. After thawing at 37℃ for 25 s, Isolate^® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.86-86
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• 2001

The aims of this work were to determine the acrosin activity and to evaluate the structural and functional integrity of AS boar spermatozoa. The acrosin activity of spermatozoa were 5.40, 4.10 and 3.40 mIU/10$^{6}$ sperm in raw, extended and frozen semen respectively , which differed significantly each other (P<0.05). After the raw and extended semen were exposured to cold and thermal shock, the acrosin activities of spermatozoa in the raw semen were 5.39, 5.21 and 5.29 mIU/10$^{6}$ sperm for control (non-shock), cold shock and thermal shock, and those of extended semen were 4.21, 3.98 and 4.00 mIU/10$^{6}$ sperm. This value among treatments did not differ significantly. The acrosin activities of spermatozoa in the extended and stored semen were 3.27, 3.52, 3.46 and 3.23 mIU/10/suup 6/ sperm, while hypo-osmotic test(HOST) values were 56.5%, 64.7%, 66.0% and 56.0%, following 4 days storage at 4$^{\circ}C$, 17$^{\circ}C$ , $25^{\circ}C$ and 37$^{\circ}C$, respectively. The results at 17$^{\circ}C$ and $25^{\circ}C$ appeared to be best compared with the other storage temperatures.