• 한국수정란이식학회지
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• 제11권3호
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• pp.309-316
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• 1996

The aim of this study is to elucidate sperm chemotaxis and to set up the optirnal condition for selection of motile and capacitated sperm from hovine frozen-semen. Thus, the effects of semen-washing after thawing, concentrations of progesterone (P4) and bovine serum albumin (BSA), and sperm-washing frequency on sperm selection were examined. For evaluating their effects, number, viability and acrosome reaction of sperm swim-up seperated from semen, which were incubated for 30 minutes at 36$^{\circ}C$ in the M2 solution containing P4 and BSA, were investigated. For frozen-semen just after thawing, sperm recovery and viability were not significantly different between P4-treated and -untreated semen. However, washing frozen-semen decreased the number of sperm and increased the viability of sperm that were recovered from semen treated with P4. Progesterone affected the recovery rate, the viability and the acrosome-reaction rate of sperm recovered from washed frozen-semen. Especially, number of motile and capacitated sperm were highest in semen treated with 50$\mu$g /ml among 0, 20, 50 and 100$\mu$g /ml of P4 concentrations. BSA affected the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4. Especially, the percentage of viable sperm were highest in semen treated with 4mg /ml among 0, 2, 4, and 6mg /ml of BSA concentrations. Repeatedly sperm-washing did not affect the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4 and 4mg /ml of BSA In conclusion, using progesterone and BSA could efficiently make the selection of motile and capacitated sperm from washed frozen-semen.

• 대한수의학회지
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• 제24권2호
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• pp.245-266
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• 1984

As a fundamental study to increase the reproductive efficiency in cattle, highly motile sperm were separated and collected from raw semen, extended semen and frozen semen by different methods using various concentrations of bovine serum albumin or Tyrode's solution. Various characteristics and light microscopic and electron-microscopic morphology of sperm separated by different methods were compared. The results obtained were as follows; 1. The sperm separated from raw semen using bovine serum albumin showed significantly high value in motility, motile sperm count, percent of normal sperm and progressive motility, as compared with control sperm and revealed the highest sperm recovery rate when separated with 6% bovine serum albumin. 2. The sperm motility, percent of normal sperm and progressive motility of the highly motile sperm frozen after being separated from raw semen with bovine serum albumin, showed significantly high value than those of a control sperm and especially found the highest value when separated with 20% bovine serum albumin. 3. Light-microscopic percent of abnormality was significantly low in the prefrozen and postfrozen highly motile sperm separated with bovine serum albumin, as compared with control sperm. 4. Electron-microscopic finding of the highly motile sperm separated with bovine serum albumin showed low percent of deformity in the dilatation and vesiculation of cell membrane, in dilatation and density loss of acrosome than in those of control sperm. 5. It was impossible to separate the highly motile sperm from frozen semen with bovine serum albumin, but it was possible with Tyrode's solution. 6. Recovery rate of highly motile sperm from raw semen extended semen and frozen semen was the highest when the sperm pellet stood in Tyrde's solution for 80 minutes. 7. The highly motile sperm separated from raw semen, extended semen and frozen semen with Tyrode's solution showed significantly high value in motility, progressive motility and percent of normal sperm, as compared with control sperm. 8. Highly motile sperm, when separated from raw semen, extended semen and frozen semen with Tyrode's solution, showed significantly low percent of microscopic abnormality as compared with control sperm.

• 한국수정란이식학회지
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• 제10권3호
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.

• 한국수정란이식학회지
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• 제24권4호
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• pp.275-279
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• 2009

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to $10^9sperm/mL$, and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

• Animal Bioscience
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• 제36권12호
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• pp.1821-1830
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• 2023

Objective: This study investigated the effect of adding seminal plasma to frozen-thawed semen on the quality of sperm and pregnancy following insemination in dromedary camels. Methods: In experiment 1, the frozen-thawed semen from 9 collections (3 bulls) was further diluted with either the base extender or homologous seminal plasma (HSP). In the second experiment, a pooled sample of frozen-thawed semen was diluted with either seminal plasma from another three bulls. Live percentage, total and progressive motility, functional and acrosome integrity, and sperm kinematics were evaluated at 15, 60, and 120 minutes post-thawing and compared to the non-treated control. In experiment 3, frozen semen was used to inseminate camels in the following experimental groups: 1-Single insemination with double dose undiluted frozen semen (n = 9); 2-Re-insemination in 6 hours with undiluted semen (n = 13); 3-Single insemination with HSP treated sperm (n = 14). Results: Frozen-thawed sperm diluted in HSP or the non-homologous seminal plasma from Bull C indicated an improvement in all parameters after 1 hour post-thawing incubation (p<0.05). The proportion of total and progressively motile sperm did not drop significantly at 60 minutes post-thawing when diluted with the seminal plasma of Bull C (p>0.05). Double insemination with nontreated sperm and single insemination with HSP-treated sperm resulted in similar pregnancy rates (15.3% vs 21.4%, p>0.05). None of the camels conceived with double-dose single insemination of nontreated sperm. Conclusion: Seminal plasma improves sperm longevity and motility after thawing in dromedary camel with a significant between-bull variation in effect. Low post-thaw sperm longevity might be the cause behind the low pregnancy rates in frozen semen insemination of dromedary camels.

• 한국가축번식학회지
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• 제26권4호
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• pp.339-345
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• 2002

The present study was carried out to investigate the effects of season on semen characteristics, frozen-thawed sperm viability and testosterone concentration in Yorkshire boars. There were no significant differences in the semen volume and sperm concentration on Yorkshire boars among spring, summer, autumn and winter. However, the pH of sperm-rich and sperm-poor fractions in winter season was higher than in spring, summer and autumn season in Yorkshlre boars. Sperm motiliy and normal acrosome of raw semen in Yorkshire boars did not differ significantly among spring, summer, autumn and winter. However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer, autumn and winter. Serum testosterone concentrations in Yorkshire were higher in spring than summer, autumn and winter. In conclusion, we found out that serum testosterone concentrations were very important role for frozen-thawed sperm viability in Yorkshire boars.

• 한국가축번식학회지
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• 제10권1호
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• 한국동물위생학회지
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• 제18권3호
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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.

• Asian-Australasian Journal of Animal Sciences
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• 제15권4호
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• pp.500-503
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• 2002

This study was carried out to investigate the effects of season influencing semen characteristics, frozen-thawed sperm viability and testosterone concentration in Duroc boars. There were no significant differences in the semen volume and sperm concentration of Duroc boars among spring, summer, autumn and winter. However, the pH of sperm-rich and sperm-poor fractions in autumn and winter season was higher than in spring and summer season in Duroc boars. Sperm motility and normal acrosome of raw semen in Duroc boars did not differ significantly among spring, summer, autumn and winter. However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer, autumn and winter. Serum testosterone concentrations in Duroc were higher in spring than summer, autumn and winter. In conclusion, when serum testosterone concentrations were higher in seasons, frozen-thawed sperm viability in Duroc boars were higher.

• 한국가축번식학회지
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• 제14권4호
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• pp.245-252
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• 1990

This study was carried out to investigate the factors affecting fertilization in vitro of follicular oocytes with frozen-thawed spermatozoa in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM-199 containing FCS and hormones. The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution. The effects of dilution and fertilization media, capacitating method, concentration of inseminated sperm and time after insemination of fertilization, were observed. The results obtained are summarized as follows : 1. The fertilization rate of frozen-thawed sperm inseminated in BO solution with caffeine and heparin together(56.4%) was higher than that of sperm inseminated in BO solution with either caffeine(10.5%) or heparin(8.9%) and without both caffeine and heparin(0%)(P<0.05). 2. The fertilization rate(56.3%) of frozen-thawed sperm inseminated in BO solution with both caffeine and heparin without preincubation was higher than that of sperm preincubated(2.9%)(P<0.05). 3. The fertilization with high concentration of frozen-thawed sperm(1.4~1.8$\times$107cells/ml) in BO solution containing caffeine and heparin resulted in higher fertilization rate, 76.7%, than the low concentration of sperm(0.8~1.0$\times$107cells/ml), 32.7%(P<0.01). 4. When the oocytes were inseminated with frozen-thawed sperm in BO solution containing caffeine and heparin without preincubation, fertilization rate increased by time and the rates were 5.9, 46.0 and 59.4% at 8, 16 and 24 hours, respectively.