• Reproductive and Developmental Biology
• /
• v.40 no.4
• /
• pp.33-37
• /
• 2016

This study was conducted to evaluate effect of ${\alpha}$-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

• Development and Reproduction
• /
• v.17 no.4
• /
• pp.345-351
• /
• 2013

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: $99.5{\pm}0.8%$, DMSO 7.5%: $99.5{\pm}0.7%$, and DMSO 10.0%: $99.6{\pm}0.6%$). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: $96.2{\pm}2.3%$, DMSO 7.5%: $95.3{\pm}3.6%$, 10.0%: $96.6{\pm}1.8%$) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm ($98.4%{\pm}0.5$) and DMSO concentration level of 5.0% ($97.8{\pm}0.1%$) were not significantly different. However, with 7.5% ($97.2{\pm}0.6%$) and 10.0% DMSO concentrations ($95.9{\pm}0.2%$) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; $66.8{\pm}1.8%$, HR: $82.0{\pm}12.9%$) and 2 years (FR; $78.5{\pm}14.8%$, HR: $79.3{\pm}0.6%$) cryopreserved sperm were lower than control (FR; 100%, HR: $91.1{\pm}3.6%$) and 2 days cryopreserved sperm (FR; $99.6{\pm}0.6%$, HR: $96.6{\pm}1.8%$). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

• Journal of Embryo Transfer
• /
• v.7 no.2
• /
• pp.133-136
• /
• 1992

Glass wool filtration and swim-up method resulted in inreasing to 58.3% and 62.7% of the progressive motility in frozen-thawed boar sperm, compared to 34.2% in the untreated sperm. Glass wool filtration tended to be more successful than swim-up method for the survival sfter incubation of 38.5$^{\circ}C$ for 3h. Sperm recovered by both the swim-up method and the glass wool filtration method were tested in an in vitro fertilization to determine which of the two techniques would yield sperm with high fertilizing capacity. The results indicated that there was a significantly(p

• Journal of Veterinary Clinics
• /
• v.34 no.2
• /
• pp.156-160
• /
• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• Journal of Aquaculture
• /
• v.19 no.1
• /
• pp.52-56
• /
• 2006

This study was conducted to investigate the effects of three different concentrations (6%, 8% and 10% final volume) of dimethyl sulfoxide (DMSO) on cryopreserved sperm of grass carp (Ctenopharyngodon idellus). Grass carp sperm was suspended in Kurokura extender #2 and equilibrated at $4^{\circ}C$ for 10 min. French straws (0.25 ml) of sperm were frozen from $4^{\circ}C\;to\;-4^{\circ}C$ at a rate of $4^{\circ}C\;min^{-1}$ and then ken $-4^{\circ}C\;to\;-80^{\circ}C$ at a rate of $11^{\circ}C\;min^{-1}$. The straws were kept at $-80^{\circ}C$ for 10 min and finally stored in liquid nitrogen $(-196^{\circ}C)$. The cryopreserved sperm was thawed in a water bath at $40^{\circ}C$ for 30 sec and fertilization, hatching rate and larval malformation were compared with fresh sperm (control). The fertilization rate of post-thawed sperm was comparable (from 88.21% to 94.30%) to that of fresh sperm. However, hatching rate of all frozen sperm were significantly lower (P<0.05) than that of control. Additionally, the larval abnormality rate of frozen sperm was significantly higher than that of fresh sperm. The results indicate that DMSO could affect the quality of cryopreserved sperm of grass carp, and a freezing program and a proper extender composition should be further studied.

• Journal of Embryo Transfer
• /
• v.28 no.3
• /
• pp.265-271
• /
• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.52-52
• /
• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.5
• /
• pp.612-616
• /
• 2004

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

• Journal of Embryo Transfer
• /
• v.9 no.3
• /
• pp.269-277
• /
• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes These experiments were conducted to examine the effect of sperm factor on the IVF and IVD, and the effect of coculture with somatic cells on the IVD of embryos. Although the concentration of epididymal sperm for IVF did not affect on cleavage rate, but 5 x 105 sperm/mi showed the highest cleavage rate(48.7%) and the developmental potential of IVF oocytes from this concentration was also greatly higher (P$^{\circ}C$-stored sperm for l2hrs and the cleavage rate from fresh sperm was significantly higher (P<0.05) than that from frozen sperm, but the developmental potential after IVF was slightly high from the frozen sperm. The cleavage rate of IVF oocytes cocultured with oviductal epithelial cells and cumulus cells was 76.3% and 72.9%, respectively. There was no difference between two coculture systems but this rate was significantly higher(P<0.05) than that of medium alone(42.0%).

• Reproductive and Developmental Biology
• /
• v.28 no.3
• /
• pp.155-159
• /
• 2004

This study was carried out to investigate the general characteristics and viability of sperm after freezing and thawing and the pregnancy rates after artificial insemination with thawed semen. The rates of viable sperm after slow and rapid freezing were 87.4±3.85% and 70.8±4.45%, respectively which were significantly lower than that of fresh semen control (91.7±3.45%). The mean concentration of epididymal sperm after dilution in 1.0 ml saline and. 3.0 ml extender in a various concentrations of cryoprotectants was 124.5±48.3 x 10/sup 6/ (range of 45 x 10/sup 6/ to 280 X 10/sup 6/ /ml). There was a significant difference not in the percentage of acrosome-reacted sperm, but in the percentage of capacitated sperm, between fresh and frozen-thawed epididymal semen. When frozen-thawed after diluting with tris-buffer extender containing glycerol, DMSO and ethylene glycol with concentration of 2 to 6%, the rates of epididymal sperm exposed to different cryoprotectants ranged from 14.4±4.7% to 20.7±5.8%, 17.8±5.2% to 36.5±4.9%, and 14.4±4.6% to 18.5±5.3%, respectively which were lower compare to fresh semen control. The pregnancy rate after artificial insemination with frozen semen was 70.6%, whereas that with fresh semen was 90.0% in dogs with naturally induced estrus.