• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• Journal of Veterinary Clinics
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• v.22 no.3
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• pp.228-232
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• 2005

Damaged spermatozoa are supposed to be trapped in glass wool. In the respect of this, two glass wool filtration spermatozoa groups (0.5 cm, 1 cm depth) were compared with control group to assay sperm motility, HOS values, and vital rate by CFDA/PI staining method following glass wool filtration. The motility of canine sperm extended with PBS+PVP after glass wool filtration was lower in both filtrated groups than that of the control group (p<0.01) and the same significant difference was also shown in canine semen extended with Tris buffer (p<0.01). The motility of canine sperm diluted with PBS+PVP was higher than that diluted with Tris buffer in the same experimental groups (p<0.05). The motility of control group was not significantly decreased until 2 hours immediately after extending, however, the motility of both glass wool filtrated spermatozoa were significantly decreased as time passed until 2 hours after filtration (p<0.01). At each time for assay (immediately, 30 min, 2 hours after filtration), the motility of canine sperm of control group was higher than the filtrated groups (p<0.05), whereas the motility of 0.5 cm depth group was higher than 1 cm depth group at the immediate time after filtration (p<0.05), 30 minutes later (p<0.05) with no difference at 2 hours. No difference was shown among the experimental groups in HOS values of canine sperm after glass wool filtration. The vital rate assayed by CFDA/PI staining of both filter groups was higher than the control group (p<0.05).

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.577-583
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• 2007

It is important to obtain semen with good quality for efficient fertilization and pregnancy. To obtain these semen, various methods have been developed but most of these methods are time consuming and require costly equipment. Therefore, the objective of this research is to investigate the usability of column filtration system as quick and simple method to get sperm with better quality. Ejaculates were obtained from 5 dogs and analyzed with basic quality parameters before each filtration. Sperm concentration was adjusted to $5{\times}10^7/ml$ after dilution. The experimental groups were divided into non-filtered group(control) and filtered groups(glass wool, Sephadex 5% and Sephadex 20%). Ejaculates were filtered through each filter system and assessed by recovery rate of sperm, motility, normal morphology, CFDA/PI stain and plasma membrane integrity(hypo-osmotic swelling test, HOST). The lowest recovery rate of spermatozoa was recorded in glass wool filtration group, followed by 20% Sephadex filtration group(p<0.05). There was no significant difference between control(non-filtered) and 5% Sephadex filtration poop. Also, there was no significant difference of sperm motility assessed under light microscope among experimental groups. Morphological normality of canine spermatozoa was the highest in the glass wool filtration group and the lowest in the 5% Sephadex filtration group with no significant differences versus 20% Sephadex filtration and control group, respectively(p<0.05). Viability of canine sperm assessed by CFCA/PI staining was the highest in the glass wool filtration poop with no significant difference versus the control group, and the lowest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, respectively(p<0.05). HOS values of canine sperm was the highest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, and the lowest in the control poop with no significant difference versus glass wool filtration group, respectively(p<0.05). Therefore, these results indicated that filtration treatment for extended canine sperm would be useful method to get sperm with better quality by trapping the damaged sperm, consequently filter would be physical barrier against injured or immotile sperm.

• 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 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.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$^{\circ}C$.

• 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.

• 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.