• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.41-46
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• 2010

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution I: 11% Lactose or Triladyl + egg yolk; solution II: solution I + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CIC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p<0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.85-91
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• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.2
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• pp.181-189
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• 2011

Cryopreservation protocols induce partially irreversible damage to mammalian sperm plasma membranes. Previous studies have indicated that adding cholesterol to the plasma membrane, as cholesterol-loaded-cyclodextrins, improves cryosurvival of sperm. Therefore, the purpose of this study was to determine if treating sperm of Markhoz bucks with cholesterol-loaded-cyclodextrins (CLC) (0, 0.75, 1.5, 2.25 and 3 mg/ml diluted $240{\times}10^6$ sperm/ml) in Tris-citric acid-glucose diluents with and without egg yolk (containing 5% glycerol) would improve the post-thaw sperm quality. The motion characteristics were evaluated with a Computer Assisted System Analyzer (CASA); acrosome integrity and vitality were measured with the triple-stain technique. Samples were recovered before and after freezing by means of putting straws into $37^{\circ}C$ water for 30 sec and then parameters were assessed. The results showed that the treatments significantly affected motility, progressive motility, recovery rate, curvilinear velocity, beat cross frequency, live sperm with reacted acrosome, live sperm with unreacted acrosome, dead sperm with reacted acrosorne, and dead sperm with unreacted acrosome during freezing (p<0.05). However; no significant differences were found for average path velocity, straight line velocity, amplitude of lateral head displacement, straightness and linearity (p>0.05). The best results were observed for extender containing 2.25 mg/ml ($240{\times}10^6$ sperm/ml) CLC supplemented with 2.6% egg yolk. In conclusion, the findings of this study indicate improved Markhoz sperm viability and motility following treatment in the presence of egg yolk.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1716-1721
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• 2006

The present study was conducted to observe the effect of osmolality of glycerolated TEST-yolk glycerol extenders on post-thawing sperm kinematics of ram spermatozoa of the native Malpura breed maintained in a semi-arid tropical environment. Good quality semen obtained from adult rams was pooled, split and diluted to 1,000 million spermatozoa per ml in complete TEST-yolk-glycerol extenders of 900, 1,200, 1,500 and 1,800 mOsm/kg osmolality. Diluted semen samples were loaded in 0.25 ml straws and cooled down to $-125^{\circ}C$ freezing temperature at the rate of $-25^{\circ}C$ per minute under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at $50^{\circ}C$ in a water bath for 10 seconds and sperm kinematics of the frozen-thawed spermatozoa were assessed by a computer-assisted sperm analysis technique. Osmolality of diluent had no significant effect on post-thawing % motility, % rapid, % medium and % slow moving frozen-thawed spermatozoa but significantly (p< 0.05) affected the % linearity and % straightness. The post-thawing % motility and % rapid motile spermatozoa were highest in samples extended in diluent of 1,500 mOsm/kg osmolality and lowest in 900 mOsm/kg. The curvilinear velocity of spermatozoa was significantly (p<0.05) higher for samples extended in 1,800 mOsm/kg, compared to those in 900 and 1,200 mOsm/kg, but the effect was not significantly different to those extended in diluent of 1,500 mOsm/kg osmolality. The study indicated that ram spermatozoa could tolerate a wide osmolality range for dilution in the complete TEST-yolk-glycerol extender for their cryosurvival. The highest recovery of motile spermatozoa following thawing was achieved in samples extended in the TEST-yolk-glycerol diluent of 1,500 mOsm/kg osmolality.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.

• Animal Bioscience
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• v.36 no.11
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• pp.1647-1654
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• 2023

Objective: Aging roosters typically exhibit subfertility with decreasing semen quality, furthermore Thai native roosters reared in rural areas are raised for a longer duration than their usual lifespan. The present study therefore aimed to assess the effect of selenium supplementation as an antioxidative substance in diets to improve the semen cryopreservation of aged roosters. Methods: Semen samples were collected from young (n = 20) and aged (n = 20) Thai native roosters (Pradu Hang Dum) at 36 and 105 weeks of age when starting the experiment, respectively. They were fed diets either non-supplemented or supplemented with selenium (0.75 ppm). Fresh semen quality and lipid peroxidation of fresh semen was evaluated before cryopreservation using the traditional liquid nitrogen vapor method. Post-thaw sperm quality and fertility potential were determined. Results: Advancing age is unrelated to decreasing fresh semen quality (p>0.05). However, lipid peroxidation in rooster semen depended on age, and the malondialdehyde (MDA) concentration increased in aged roosters (p<0.05). Selenium supplementation in diets significantly decreased the MDA concentration and increased the sperm concentration (p<0.05). In contrast, cryopreserved semen was affected by advancing rooster age, and selenium influenced sperm quality (p<0.05). Younger roosters had higher post-thaw sperm quality and fertility potential than aged roosters (p<0.05). Likewise, diet selenium supplements improved post-thaw sperm quality and fertility compared with the non-supplement group. Conclusion: Rooster's age does not influence the rooster sperm quality of fresh semen, while sperm cryotolerance and fertility were greater in young roosters than in aged roosters. However, sperm of aged roosters could be improved by dietary selenium supplementation.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.369-375
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• 2011

The aim of this study was to evaluate the effect of ${\alpha}$-tocopherol on blastocysts development and subsequent cryosurvival of the vitrification. The ${\alpha}$-tocopherol(0, 100, 200, 400 ${\mu}M$) was added in to culture medium for the bovine embryos. The blasocysts from the ${\alpha}$-tocopherol and untreated control groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. There were no differences in the overall cleavage rate($56.14{\pm}4.66$, $58.18{\pm}4.70$, $62.97{\pm}6.86$ and $51.17{\pm}7.28$) among four treatment groups. However, in blastocyst development and total cell number were significantly higher in ${\alpha}$-tocopherol 200 ${\mu}M$ ($38.60{\pm}7.12$; $106.33{\pm}3.50$) to culture medium than other treatment groups($29.30{\pm}5.24$, $31.60{\pm}7.12$ and $26.37{\pm}4.18$; $101.36{\pm}5.12$, $97.27{\pm}2.87$, and $91.23{\pm}7.52$ respectively). Before and after vitrification, the total cell number and blastocyst development of embryo were significantly higher in July to August than September to October. In conclusion, addition of ${\alpha}$-tocopherol 200 ${\mu}M$ to in vitro bovine embryo culture medium was beneficial for improving embryo quality by decreasing the embryo damage blsstocysts cell number and improving the tolerance of the embryos to cryopreservation.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.175-179
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• 2007

The objective of this study was to investigate the effect of thawing temperature on the sperm viability and acrosomal morphology for semen storage of miniature pig by the 0.5ml straw method. In this present study, sperm viability (SYBR-14/PI staining), membrane integrity (Hypoosmotic Swelling Test), acrosome intactness, intensity and capacitation status (chlorotetracycline staining) in frozen miniature pig sperm were evaluated after thawing at 37, 50 and $70^{\circ}C$ for 5, 10 and 45 sec, respectively. Interestingly, the results indicated that sperm thawed at $70^{\circ}C$ for 5 sec significantly (p<0.05) increased sperm viability, but lower the percentage of AR (acrosome reacted spermatozoa) pattern compared to sperm thawed at $37^{\circ}C$ for 45 sec and $50^{\circ}C$ for 10 sec. In terms of thawing condition, high temperature for a short time using the 0.5ml straw was improved cryosurvival of miniature pig semen. Therefore, appropriate thawing method for cryopreservation of miniature pig is required for increasing post-thawing viability.