• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.420-430
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• 2000

These studies were preformed to investigate the freezing conditions to achieve good post-thaw viability of spend and the practical methods of artificial insemination frozen canine semen. Semen were collected from nine male dogs which had been proved to be fertile in the past and the semen were treated for freezing procedure. Post-thaw motility and viability of canine sperm were evaluated to investigate individual tolerance of freezing, difference among freezing extenders, dif-ference among freezing equipments and freezing conditions, difference between fast and slow cooling rate, difference according to different glycerol concentration, effect of seeding on post-thaw viability, difference according to cutting part of straw, difference according to thawing temperatures, and dif-ference according to media added to thawed semen. Thawed semen for insemination were added with equal volnme of canine capacitation medium (CCM) and the volume of semen and the number per insemination were adjusted as 2-3 ml and $20-30 {\times}10^7,$ respectively. The semen were inseminated in vagina using balloon catheter and en17ryos were cellected from 9 to 11 days after the second Al to d determine fertilization.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.278-278
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• 2004

The purpose of this study was to establish the convenient freezing method for more cheap and simple. Semen quality was evaluated the motility, viability, abnormality, acrosome intactness and membrane integrity. And there were also examined the developmental rates of IVM/IVF embryos using frozen-thawed boar semen in each treatment group. (omitted)

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.13-17
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• 2011

Research in the area of equine artificial insemination (AI) has led to its increased application in field trials. However, procedures for equine semen collection, cooling and freezing of semen and artificial insemination need further improvement. In experiment 1, we investigated the percentage of total motility (TM) and progressive motility (PM) of sperms at after-collection, cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 2, mares were inseminated with either cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 3, we examined the effect of buffer (skim-milk extender), which was infused into the uterus at the time of AI with frozen-thawed semen. In experiment 4, we compared AI pregnancy rates for mares ovulating spontaneously versus after treatment with hCG. In experiment 1, the average percentage of TM was decreased from 75.3% to 14.4% at the stage of after-collection to frozen-thawed semen (p<0.05). The average percentage of PM was 58.2% and 59.6% at after-collection and cooled-diluted, but it was significantly increased 71.7% after frozen-thawed (p<0.05). In experiment 2, the pregnancy rates after AI using cooled-diluted, cooled-transported and frozen-thawed semen were 60%, 50% and 37.5%, respectively, and similar among treatments. In experiment 3, the pregnancy rate of mares infused with buffer at AI was 40% which was higher than that with no buffer (10%). In experiment 4, the pregnancy rates of mares were similar between ovulated spontaneously (25%) and ovulated with hCG (50%). The results suggest that equine semen that has been cooled-diluted, cooled-transported or frozen can be successfully used to establish AI, pregnancy and foal production. Also, the pregnancy rates after AI can be increased by infusing buffer into the uterus at AI or by inducing ovulation with hCG, but further study is need.

• 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.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.155-159
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• 2004

This study was carried out to investigate the general characteristics and viability of sperm after freezing and thawing and the pregnancy rates after artificial insemination with thawed semen. The rates of viable sperm after slow and rapid freezing were 87.4±3.85% and 70.8±4.45%, respectively which were significantly lower than that of fresh semen control (91.7±3.45%). The mean concentration of epididymal sperm after dilution in 1.0 ml saline and. 3.0 ml extender in a various concentrations of cryoprotectants was 124.5±48.3 x 10/sup 6/ (range of 45 x 10/sup 6/ to 280 X 10/sup 6/ /ml). There was a significant difference not in the percentage of acrosome-reacted sperm, but in the percentage of capacitated sperm, between fresh and frozen-thawed epididymal semen. When frozen-thawed after diluting with tris-buffer extender containing glycerol, DMSO and ethylene glycol with concentration of 2 to 6%, the rates of epididymal sperm exposed to different cryoprotectants ranged from 14.4±4.7% to 20.7±5.8%, 17.8±5.2% to 36.5±4.9%, and 14.4±4.6% to 18.5±5.3%, respectively which were lower compare to fresh semen control. The pregnancy rate after artificial insemination with frozen semen was 70.6%, whereas that with fresh semen was 90.0% in dogs with naturally induced estrus.

• 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.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.271-274
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• 2010

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.1-8
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• 2005

This experiment was carried out to investigate the effects of saccharide in the lactose-egg yolk(LEY) extender for freezing of boar semen on the viability, normal acrosome, fertilizable of in vitro or in vivo oocyte after thawed. Normal acrosome post-thawed spermatozoa was higher when increasing of glucose concentration in LEY extender with 3 or $4\%$ glycerol, but viability was not significant. Viability of the post-thawed spermatozoa was higher when fructose or fructose and glucose were added to LEY extender with $3\%$ glycerol than glucose and sucrose or fructose, glucose and sucrose(P<0.05). Rate of normal acrosome of post thawed spermatozoa was higher when both fructose and glucose$(81.4{\pm}2.3\%)$ were added to the LEY extender than saccharide not added$(41.6\pm0.6\%)$ to it(P<0.001). The percentage of fertilization, cleavage and development to blastocyst of oocytes fertilized with post-thawed spermatozoa from freezing by LEY extender were $70.8\~80.7\%$, $44.6\~45.7$ and $13.6\~16.0\%$, respectively. Conception rate by artificial insemination with frozen boa. semen was higher$(83.1{\pm}0.3\%)$ than commercial frozen semen from SGI company$(50.0{\pm}0.1\%,\;P<0.05)$, but litter size were no significant differences between frozen by LEY extender$(9.4{\pm}1.7\~10.4{\pm}0.7head/sow)$ and SGI semen$(8.0{\pm}1.1 head/sow)$.

• 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.