• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.201-206
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• 2001

This study was carried out to investigate the effects of spring (March~May) and summer (June~August) influencing semen characteristics, frozen-thawed sperm viability and serum testosterone concentration in Yorkshire boars. Results of this study were as follows: 1. There were no significant differences in the semen volume, pH and sperm concentration of sperm-poor fraction of Yorkshire boars between spring and summer. However, sperm concentrations of sperm-rich fractions in spring were higher than those in summer (P＜0.05). 2. Sperm motility and normal acrosome of raw semen in Yorkshire boars did not differ significantly between spring and summer, However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer season (P＜0.05). 3. Serum testosterone concentrations in Yorkshire boars were 4.04 ng/$m\ell$ in spring and 2.85 ng/$m\ell$ in summer. Serum testosterone concentrations in spring were higher than those in summer (P＜0.05). 4. In conclusion, when serum testosterone concentrations in Yorkshire boars were higher, frozen-thawed sperm viability was higher.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.115-118
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• 2008

Glycerol is the cryoprotectant most frequently used to freeze semen in several of species. The objective of the present study was to compare the effect of three different glycerol concentrations (4, 6 or 8%, v/v) on frozen-thawed dog sperm survival rate. Ejaculates from 9 dogs collected by digital manipulation were pooled and assessed by macroscopic and microscopic criteria. Semen was divided into 3 aliquots, which were centrifuged and the sperm pellets rediluted with first Tris-glucose-citric acid extender. After 1 h cooling at $4^{\circ}C$, second extender containing 4, 6 or 8% glycerol was added, respectively. The semen was loaded into 0.25 ml straws and frozen and stored in liquid nitrogen and thawed. Sperm vigor, live:dead spermatozoa ratio using HOS test, and sperm morphology using $Spermac^{(R)}$ stain were evaluated. After thawing, there were no significant differences among groups in vigor, viability and morphology. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen. Therefore the use of 4% glycerol in the extender has less toxic effect and reduces of freezing injuries.

• 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.

• 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.23 no.4
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• pp.239-243
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• 2008

This study was conducted to investigate the effects of type of the sugar supplemented to the extender on the vigor, viability and intact acrosomal rates of frozen-thawed dog spermatozoa. The ejaculated semen was diluted with TRIS-citric acid extender containing 200mM TRIS, 73mM citric acid, 6% (v/v) glycerol, 20% (v/v) egg yolk, 1% (v/v) antibiotics (streptomycin/penicillin), 44 mM sugar, which was either glucose, fructose or glucose-fructose combination, and distilled water to make the final volume of 100ml. Extended semen samples were cooled at $4^{\circ}C$ for an hour, packaged in 0.25ml straws, equilibrated for 10 minutes in liquid nitrogen vapor, and frozen in liquid nitrogen. Samples were thawed by placing straws into $37^{\circ}C$ water for 120 seconds. After thawing, vigor, viability and intact acrosomal rates of frozen-thawed semen were compared according to type of sugar. No significant differences were observed between glucose and fructose groups. In addition, combination of the 2 sugars also did not show any significant differences in the vigor, viability and intact acrosomal rates. In conclusion, glucose and fructose were equally efficient as sugar supplements for freezing extender.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.898-908
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• 1998

The aim of this experiments were to assess the time-interval change of motional characteristics in frozen-thawed semen of Korean native cattle (KNC) by using computer aided semen analysis (CASA) technology. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system (SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber (Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The measurement parameters in SIAS were as follows ; frame rate = 30 frames per sec, image capture = 1 sec, minimum motile speed = $10{\mu}m/s$, maximum countable sperm number = 400. Statistical analysis was done by Student t-test with use of the Sigma plot program on a IBM personal computer. The dancemean(DNM) and hyperactivated sperm(HYP) of frozen-thawed KNC semen kinematics were significantly decreased(p < 0.05) after 10 min of incubation at $37^{\circ}C$ water bath. But, wobble(WOB) of same sample semen was significantly increased(p < 0.05) after 10 min of incubation and significantly decrease(p < 0.05) after 60 min of same incubation. And, after 30 mim of incubation, significantly differences were found most of motion kinematics, motifity(MOT), curvilinear velocity(VCL), straight line velocity(VSL), average path velocity(VAP), amplitude of lateral head displacement(ALH), beat cross frequency(BCF), mean angular displacement(MAD), dance(DNC), on same sample semen. The DNM of KNC semen sample was variable kinematics after 30 min of incubation. Also, the linearity(LIN) and straightness(STR) was significantly decreased(p < 0.05) from 60 min of incubation. In conclusion, the AI within 30 min after thawing of frozen semen can be an effective method for obtaining high fertility rate in KNC reproductive program.

• Journal of Veterinary Clinics
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• v.18 no.1
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• pp.44-47
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• 2001

Semen were collected from 9 male dogs and frozen by liquid nitrogengas. Frozen semen were thawed at 7$0^{\circ}C$ for 8 seconds. About $2{\timss}10^8$ sperm per insemination were inseminated to 10 bitches (3 Retrievers, 4 Chihuahuas, 1 Yorkshire Terriers, 1 Maltese, and 1 Poodle) at three and six days after the estimated peak of luteinizing hormone. For small breed dogs, uretero-renoscope (Kahl Storz, Germany, 12.5 Fr) was used for trans-cervical insemination, whereas cystoscope(Kahl Storz, Germany, 22Fr) was used for large breeds (Retrievers). Pregnancy was diagnosed by ultrasonography at 30 days after insemination. All of 3 Retrievers (100.0%) and 3 bitches of 7 small breed dogs (42.9%) were conceived (60.0% in total). This result indicated that trans-cervical insemination using endoscope is an effective method for AI with frozen semen not only for large breed dogs such as Retriever but also for small breeds.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.612-616
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• 2004

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.190-197
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• 2008

This study was designed to analyze boar sperm to compare motility, acrosome morphology, viability and ATP by various preservation methods between Duroc boar A with cold shock resistance sperm and Duroc boar B with cold shock sensitivity sperm. Semen volume, sperm concentration, motility and normal acrosome between Duroc boar A and B did not show any differences within 2 h after collection. There were no differences in sperm motility and normal acrosome between boar A and B at 1 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively. However, sperm motility and normal acrosome from 2 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively, were higher for boar A than boar B. The frozen-thawed sperm motility and normal acrosome were higher for boar A than boar B. The sperm viability and ATP concentration according to storage period of liquid semen at $17^{\circ}C$ and $4^{\circ}C$ were higher for boar A than boar B. Also, the sperm viability and ATP concentration of frozen-thawed semen were higher for boar A than boar B. In conclusion, we found out that the original quality of boar semen with cold shock resistance sperm played an important role.

• 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$(8.0{\pm}1.1 head/sow)$.