• Journal of Animal Science and Technology
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• v.47 no.6
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• pp.925-936
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• 2005

The present study evaluated whether pentoxifylline added to the freezing extender could improve the sperm characteristics and function in canine frozen semen. Also the conception rate following AI with frozen-thawed semen was investigated. The beneficial effects of pentoxifylline supplementation were visible in motility, viability, acrosome reaction, and tail swelling patterns. Especially, highest sperm viability and function were obtained in the forzen semen supplemented with 1mM pentoxifylline. The follicle size measured by ultrasonography was 6.48 mm, 11.52 mm and 8.9 mm on 11, 13 and 15 days after the onset of natural estrus, respectively and ovulation occurred on 13 and 15 days. The pregnancy rates in bitches inseminated with frozen semen on natural and induced estrus were 71.4% and 75.0%, respectively. There was no significant difference between the pregnancy rates in bitches inseminated with frozen semen following natural and induced estrus, but the litter size was slightly increased in natural cycle.

• 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 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.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.

• 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$^{\circ}C$.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.90-90
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• 2002

The purpose of this study was to investigate the effect of kind and combination of sugars on the viability and acrosome damage of post-thaw spermatozoa in canine. The extender used was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as single (fructose, xylose, trehalose), two combinations (Fruc+Tre, Fruc+xyl, Tre+xyl) and three combinations (Fruc+Tre+Xyl). The concentration of sperm collected were adjusted of 50${\times}$10$\^$6/ per straw for freezing. The frozen spermatozoa were thawed at 37$^{\circ}C$ for 1 min and then analysis for CASA program in Livestock Improvement Main Center, NACF. The motility of post-thaw spermatozoa in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (79％ vs. 63, 66, 70, 71, 74 and 75%). The progressive motility after CASA analysis in Fuc+Tre group was also higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (67% vs. 53, 57, 60, 61, 62 and 64%). The acrosome damage of post-thaw spermatozoa stained was not significantly different among treatment groups such as fructose, trehalose, xylose, Fru+Tre, Fru+xyl, Tre+xyl and Freu+tre+xyl (17.7, 18.3, 28.0, 17.0, 19.7, 20.0 and 19.0%). The results indicated that the motility and progressive motility of post-thaw spermatozoa in Fru+Tre group was better, and acrosome normality was not different among all groups. The use of Tris-buffer supplemented with Fru+Tre as sugar for frezing of canine spermatosoa could be better and apply to semen banking and artificial insemination.

• 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.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.195-201
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• 2003

This study was carried out to establish most suitable freezing condition, to evaluate the different condition of freezing and thawing rates on the viability and motility of frozen canine spermatozoa. The ejaculated semen was added to obtained 200∼400 ${\times}$ 10$^{6}$ /$m\ell$ with extender I and was cooled to 4$^{\circ}C$ over 30, 60 and 120 minutes. And then, semen was diluted with extender II containing 4, 6 and 8％(v/v) glycerol for 60 min, respectively and packaged in 0.5$m\ell$ straw, equilibrated far 30, 60 and 120 min at 4$^{\circ}C$ and cryopreserved in liquid nitrogen vapor at different distance(3, 5, 7 and 9 cm, respectively), plunged into nitrogen tank. Samples were thawed by placing straws into 27, 37, 47, 57$^{\circ}C$ water bath for 120, 20 and 12 sec, respectively. The results were as follows; 1. The survival and motility rate immediately post-thawing was significantly higher in samples frozen in 4％ glycerol than 6 or 8％ glycerol(P< 0.05). 2. According to equilibration time at 4$^{\circ}C$, the survival and motility rate immediately post-thawing was significantly higher in samples frozen after 60 min equilibration than 30 or 120 min equilibration(P<0.05). 3. Freezing in distance of 5 cm from liquid nitrogen yield better survival and motility rate than the others(3, 7 or 9 cm)(P<0.05). 4. The effect of thawing rate on sperm survival were higher when the thawing was done at 37$^{\circ}C$ for 120 sec(P<0.05).

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.486-492
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• 2007

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.137-143
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• 2002

This study was conducted to evaluate the effect of sugar kinds and combination of various sugars, and straw size and pre-freezing time on motility of post-thaw spermatozoa in canine. In general, the extender was supplemented with fructose or glucose for semen freezing. The extender used in .this study was Tris-citric acid extender (Tris-buffer) supplemented with 20％ Egg-yolk, 8％ glycerol, 1％ Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer. were combined such as control (fructose, xylose, trehalose), two combinations (Fru＋Tre, Fru＋Xy1, Tre＋Xy1) and three combinations (Fru＋Tre＋Xy1). The motility after CASA analysis in Fru＋Tre＋Xy1 was higher than those in fructose, trehalose, xylose, Fru＋Tre, Fru＋Xy1, Tre＋Xy1 (69 vs. 58, 61, 50, 65, 20, 54). However, the progressive motility after CASA analysis in Fru＋Tre group was higher than those in fructose, trehalose, xylose, Fru＋Xy1, Tre＋Xy1, Fru＋Tre＋Xy1 (59％ vs. 47, 55, 42, 13, 49, 44％). The survival rate of post-thaw spermatozoa in 0.25 $m\ell$ straw for 10 min pre-freezing was significantly higher than those in 0.25 $m\ell$ straw for 10 min, 0.25 and 0.5 $m\ell$ for 5 min (80＋0.0 vs. 65＋7, 68＋16, 58＋8％; P＜0.05). The results indicated that the progressive motility of post-thaw spermatozoa in 70 mM Fru ＋Tre (two combination) following CASA analysis and 0.25 ml straw for 10 min pre-freezing time could be better for freezing of semen in canine.