• Korean Journal of Veterinary Research
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• v.33 no.2
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• pp.337-343
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• 1993

To determine the test conditions to enhance the use of hamster test in dogs, semen were collected from four dogs which had been proven to be fertile in the past and then preserved in BWW (Biggers, Whitten, Whittingham) medium for about 20 hours. The semen were given each different treatment according to the experimental design and coincubated with zona-free hamster ova for 5 hours. The ova were stained by lacmoid and examined under phase contrast microscope to investigate the rates of ova bound with sperm(sperm binding) and ova penetrated by sperm (penetration), and also numbers of both bound and penetrated sperm per ovum. In comparison of different concentrations of canine sperm, the rate of sperm binding was higher in $1.5{\times}10^8$, $1{\times}10^8$, and $5{\times}10^7$ sperm concentrations than $5{\times}10^5$ concentration(p<0.01), and also than $5{\times}10^6$ concentration(p<0.05), respectively. The number of bound sperm per ovum was considerably higher in $1.5{\times}10^8$ sperm concentration than $5{\times}10^7$, $1.5{\times}10^6$, and $5{\times}10^5$ concentrations(p<0.01). The rate of penetration was considerably higher in $1.5{\times}10^8$ and $1{\times}10^8$ sperm concentrations than $5{\times}10^5$ concentration,(p<0.01), and also the higher result of penetration was shown in $5{\times}10^7$ than $5{\times}10^5$ (p<0.05). The number of penetrated sperm per ovum was considerably higher in $1.5{\times}10^8$ sperm concentrations than $5{\times}10^5$(p<0.01), and also the higher number was shown in $1{\times}10^8$ than $5{\times}10^5$ (p<0.05). In comparison of the different preincubation period of canine spermatozoa, no difference was obtained in the results of hamster test among the preincubation periods of 4 hours, 18~24 hours and 48 hours. The canine spermatozoa in BWW medium with $Ca^{2+}$(1.3mM) and without FCS(fetal calf serum), with both $Ca^{2+}$(1.3mM) and FCS, with $Ca^{2+}$(2.6mM) and without FCS, and with both $Ca^{2+}$(2.6mM) and FCS showed no difference in the results of hamster test.These results indicated that the appropriate concentration of sperm should be given in hamster test for dog sperm.

• 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.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• Journal of Veterinary Clinics
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• v.28 no.6
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• pp.571-575
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• 2011

Sperm cryopreservation methods have been improved over the last few decades. However, an optimized thawing rate has not yet been established. Therefore, we investigated the effect of thawing rate on sperm function after cryopreservation. The ejaculates collected from beagle dogs were cryopreserved and then thawed at two different thawing rates ($37^{\circ}C$ for 1 min or $70^{\circ}C$ for 15 sec). The thawed sperm were evaluated for motility, viability, morphology, plasma membrane integrity, phosphatidylserine (PS) translocation, and intracellular $H_2O_2$ level. The sperm thawed rapidly at $70^{\circ}C$ showed improved motility, viability, normal morphology, plasma-membrane integrity and non-PS translocation compared to the sperm thawed slowly at $37^{\circ}C$ (P < 0.05). However, the intracellular $H_2O_2$ levels were not significantly different between the rapid- and slow-thawed sperm (P > 0.05). In conclusion, sperm rapid thawing at $70^{\circ}C$ could improve the function of cryopreserved canine sperm, and the appropriate thawing rate would enhance the quality of the cryopreserved sperm.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2009.04a
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• pp.304-304
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• 2009

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.5
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• pp.646-651
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• 2016

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

• Korean Journal of Veterinary Research
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• v.34 no.4
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• pp.851-855
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• 1994

The semen from four male dogs which had been proven to be fertile in the past were frozen in a deep freezer at $-60^{\circ}C$ by simple freezing method using methanol and preserved at the same teperature for from 7 to 10 days. The semen were inseminated to 7 female dogs in estrus to find out the usability of this freezing method in artificial insemination for dogs. In addition, post-thaw motility and viability of sperm from two male dogs which had been fertile were also evaluated to investigate individual difference. Successful pregnancy was obtained by artificial insemination with canine semen frozen at $-60^{\circ}C$ by simple freezing method using methanol, namely, 3 bitches among 7 bitches which had been inseminated delivered puppies(42.8%). The average litter size of the whelping dogs were 4.3 puppies. The average post-thaw motility of canine sperm in the cases of conception was showen higher than those of non-conception(65.0% vs. 42.5%), along with the, same result in the average post-thaw viability between the two groups(53.3% vs, 27.5%). Individual difference of post-thaw motility and viability was obtained between two fertile dogs(p<0.05).

• 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.

• Journal of Veterinary Clinics
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• v.8 no.2
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• pp.183-190
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• 1991

Pellet and straw methods in canine semen freezing are compared with respect to motility, viability and acrosome demage of sperm during each of the two major processing steps, to prior-freezing and to frozen-thawing. Senen was extended with a tris-buffered egg yolk contained 4% glycero1 Pellet freezing in the hole of dry ice and straw freezing on the surface of liquid nitrogen were carried out, respectively. The frozen semen 10 days after storage in liquid nitrogen container. wao thawed. In the comparison of two freezing methods, the straw freezing method with 42.7% in motility. 49.2% in viability and 0.186 acrosome score after thawing seems to be superior to the pellet freezing method with 31.2%, 34.5% and 0.314%, respectively. Sperm motility of processing step to frozen-thawing against decrease rate 12.67% to Prior freezing appeared of 33.84% and 49.37% in straw and pellet freezing and increase of 0.02 in acrsomal score to prior freezing appeared of 0.08 and 0.21 in straw and pellet freezing method to frozen-thawing

• Journal of Veterinary Clinics
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• v.16 no.2
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• pp.375-380
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• 1999

This study was conducted to develop an intrauterine inseminator (IUI) to deposit of frozen semen into uterus and to evaluate the results obtained after artificial insemination by IUI. Two Japanese spitzs (2 to 4 years of age) were used as semen donors. Semen was collected by manual masturbation into sterile glass collection tubes and separated into 3 fractions with only the sperm-rich fractions retained for further examination. Sperm motility >70%, sperm concentration of 200 to $400{\times}10^6 cells/ml$$\times$g for 5 min and poured out the suspended solution, and then diluted with 2 ml Tris-buffer which was consisted of 2.4 g Tris, 1.4 g citric acid, 0.8 g glucose, 0.1 $\mu\textrm{g}$/ml streptomycin, 100 IU/ml penicillin, 20 ml egg yolk to 100 ml mili-Q water (Ext I) or supplemented with 8 ml glycerol and 1 ml Equex STM paste to 100 rnl (Ext II). The diluted semen was cooled to 5$^{\circ}C$ in cold room, where the temperature in the sample reached 5$^{\circ}C$. Two h after beginning the cooling procedure, 2 ml of Ext II, also at 5$^{\circ}C$, was added and mixed by gently reversing the tubes several times during 1 h. The final sperm concentration for freezing was approximately $50{\times}10^6 cells/ml$. After equilibration, the semen was loaded into 0.5 ml straw and frozen on the liquid nitrogen vapour in styrofoam box. The straws were thawed at 7$0^{\circ}C$ for precisely 6 sec. After thawing of each straw, the frozen semen can survived over 50% motility. All the females were inseminated twice with 1 ml of $25{\times}10^6 cells/ml$