• Korean Journal of Veterinary Service
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• v.18 no.3
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• pp.1-12
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• 1995

To investigate effect of seeding on post-thaw motility and viability of canine spermatozoa, the semen from male dogs which had been proved to be fertile in the past were frozen and seeded during freezing process. Post-thaw motility and viability of canine sperm which were frozen and seeded were investigated according to different seeding temperatures of $-5^{\circ}C$, $-10^{\circ}C$, or $-l5^{\circ}C$ and also according to different concentration of glycerol of 2%, 5% and 10%. In addition, post-thaw motility of canine sperm frozen by direct freezing in a deep freezer or programmed freezing in a programmed cell freezer was investigated. Post-thaw motility of canine sperm was compared according to different seeding temperatures : The sperm seeded at $-5^{\circ}C$ showed considerably higher post-thaw motility than that of non-seeding, and that seeded at $-10^{\circ}C$, or $-l5^{\circ}C$, respectively, in 2% and 5% glycerol groups on both 2 and 7day after freezing(p<0.05). In 10% concentration of glycerol, the sperm seeded at each seeding temperature showed considerably higher post-thaw motility than that of non-seeding group on day 7 after freezing(p<0.01). Post-thaw viability of canine sperm was compared according to different seeding temperatures : The sperm seeded at $-5^{\circ}$ showed significantly higher post-thaw motility than that of non-seeding, and that seeded at $-10^{\circ}C$, or $-l5^{\circ}C$, in 5% and 10% glycerol groups on day 7 after freezing(p< 0.05). In comparison of post-thaw motility of canine sperm seeded according to different concentration of glycerol, 5% glycerol group and 10% glycerol group showed considerably higher post-thaw motility than 2% glycerol group without difference between those two groups in all seeding temperatures($-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}C$) on day 2 and 7 after freezing(p<0.01). In comparison of post-thaw viability of canine sperm seeded according to different concentration of glycerol, 5% glycerol group and 10% glycerol group showed the same considerably higher post-thaw viability than 2% glycerol group on each thawing day(p<0.01). The canine sperm frozen and seeded by programmed freezing method showed considerably higher post-thaw motility than that frozen by direct freezing method in all different seeding temperatures($-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}$). These results indicated that the higher post-thaw motility and viability was obtained in the spermatozoa seeded than that of non-seeding, that among different seeding temperatures of $-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}C$, the sperm seeded at $-5^{\circ}C$ showed higher post-thaw motility and viability than the other temperatures, also among different concentrations fof glycerol of 2%, 5% and 10%, the sperm frozen and seeded in 5% and 10% concentration of glycerol showed higher post-thaw motility and viability than that in 2% of glycerol, and that the sperm frozen and seeded by programmed freezing method showed higher motility than that by direct freezing method.

• 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.19 no.1
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• pp.80-85
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• 2002

This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.

• 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 Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.268-272
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• 2020

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSC-exo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 ㎍/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 ㎍/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.256-256
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• 2004

The study was carried out to investigate the effects of preservation of ovaries and oocytes with and without cumulus cells and incubation time on zona penetration by canine spermatozoa. The objective of this study was to produce in vitro fertilized oocytes and solute canine sterile. (omitted)

• 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.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• 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.

• Journal of Veterinary Clinics
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• v.10 no.1
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• pp.1-10
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• 1993

To investigate the availability of hamster test in assaying the fertilizing capacity of dog sperm and the effect of canine sperm motility on sperm binding and penetration, semen were collected from four dogs(three dogs had been proven to be fertile and one dog to be subfertile during the past two years) 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 between fertile dogs and a subfertile dog, the rate of sperm binding was higher in fertile dogs than the subfertile dog(p<0.01, p<0.05). The number of bound sperm per ovum was considerably higher in a fertile dog than the subfertile dog((p<0.01), and also difference of number of the bound sperm was obtained among the fertile dogs(p<0.01, p<0.05). The rate of penetration as well as the number of penetrated sperm per ovum was higher in the fertile dogs than the subfertile dog(p<0.01, p<0.05). In fertile dogs. the canine semen preserved at 4$^{\circ}C$ for 18 to 22 hours showed from 30 to 80% motility at Insemination, however, no difference in hamster test was obtained according to different degree of sperm motility. These results indicated that hamster test would be of avail in assaying the fertilizing capacity of dog sperm.