• Asian-Australasian Journal of Animal Sciences
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• 제13권6호
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• pp.739-747
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• 2000

This experiment was conducted to evaluate the influence of dimethyl-sulfoxise (DMSO, 0, 5, 50, 100 and $500{\mu}M$) on the motility and acrosome reaction of the frozen-thawed spermatozoa from 3 different bulls (Bull A, Band C). Also we evaluated the developmental capacity of bovine embryos fertilized in a medium containing DMSO at various concentrations. DMSO had negligible effects on the sperm motility and acrosome reaction in all three bulls. However, the development rates from 2 to 16 cells stage on the 3rd day after insemination with 50, 100 and $500{\mu}M$ DMSO in Bull-B, and up to the blastocyst stage fertilized with 5, 50, 100 and $500{\mu}M$ in Bull-A were significantly higher (p<0.05) than those of control ($0{\mu}M$ DMSO) group from each bull. Furthermore, the rates of blastocysts per cleaved embryos of 5 to $500{\mu}M$ DMSO group in Bull-A and of 5 to $100{\mu}M$ DMSO in Bull-C were also significantly higher (p<0.05) than those for their $0{\mu}M$ groups, respectively. These results indicate that DMSO at micromol level used for in vitro fertilization might stimulate the development of embryos for some bulls.

• 한국가축번식학회지
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• 제10권1호
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• pp.91-99
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• 1986

This experiment was undertaken to examine the effects of HIS treatment on the motility and acrosome reaction of frozen bovine spermatozoa and to test their abilities to interact with zona-free hamster eggs in vitro. Also, in vitro results were compared with those of bull's fertility in AI. The frozen semen from four Holstein bulls were exposed to HIS-DM for 5 minutes after thawing and then preincubated for 60 minutes in DM prior to insemination. The hamster eggs were mounted, fixed and stained 6 hours after exposure to boving spermatozoa and examined under a phase-contrast microscope. 1. The sperm motility expressed as a mobility index dro, pp.d significantly from 60-75 to 12-24 after exposure to HIS-DM, but increased in 32 to 41 at insemination. Bull C showed a low motility index than those of the orher bulls. The percentage of acrosome reaction by staining procedure were increased by HIS-DM treatment but did not change during 7 hours incubation period in DM. 2. The overall percentage of hamster eggs interacting with bull spermatozoa was 56.3%, 58.3%, 66.6% and 70.0%, respectively. Although there was no significant difference among bulls in the penetration rate of spermatozoa into hamster eggs, high proportions of eggs interacted with spermatozoa from Bull C and D than those from Bull A and B. 3. The conception rates (60-90 day RP) resulting from AI were 62.5%, 67.5% and 70.9% for Bull A, B and C, respectively. These results were in good agreement with the invitro results that the proportions of bull sperm-egg interction were greater for Bull C than for Bull A and B.

• 한국가축번식학회지
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• 제14권2호
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• pp.101-106
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• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Clinical and Experimental Reproductive Medicine
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• 제12권2호
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• pp.99-116
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• 1985

소의 번식효율을 증가시키기 위한 기초실험으로서 6%, 10% 및 20%의 bovine serum albumin 을 사용한 선인들의 방법과 tyrode액을 사용하여 시도한 저자의 방법으로 소의 원정액, 희석정액 및 일반시판 냉동정액으로부터 고활력정자를 분리수집하여 정자의 각종 성상과 광학현미경적 형태를 비교관찰한 결과는 다음과 같다. 1. 원정액으로부터 bovine serum albumin을 사용하여 분리한 정자는 대조군에 비하여 운동성, 운동성 정자수, 정상정자율 및 전진운동성이 현저히 높았고 정자회수율은 6%일 때 가장 높았다. 2. 원정액으로부터 bovine serum albumin을 사용하여 고활력정자를 분리한 후 냉동한 정액의 정자운동성, 정상정자율 및 전진운동성은 대조군에 비하여 현저히 높았고, 이러한 현상은 bovine serum albumin의 농도가 20%일 때에 가장 현저하게 나타났다. 3. Bovine serum albumin을 사용하여 분리한 고활력정자의 냉동전 및 냉동후의 광학현미경적인 기형율은 대조군에 비하여 현저히 낮았다. 4. Bovine serum albumin을 사용하여 분리한 고활력정자는 전자현미경으로 세포막의 확장 및 공포 형성, acrosome의 확장과 density loss의 변형율이 대조군에 비하여 낮았다. 5. 일반시판 냉동정액으로부터의 고활력정자의 분리는 bovine serum albumin을 사용할 때는 어려웠으나, tyrode액을 사용한 이 실험에서는 가능하였다. 6. 원정액, 희석정액 및 냉동정액의 고활력정자회수율은 tyrode 액을 이용하여 80분간 정치하였을때 현저히 높았다. 7. Tyrode 액을 이용하여 원정액, 희석정액 및 냉동정액으로부터 분리된 고활력정자의 운동성, 전진운동성 및 정상정자율은 대조군에 비하여 현저히 높았다. 8. Tyrode 액을 이용하여 원정액, 희석정액 및 냉동정액으로부터 분리한 고활력정자의 광학현미경적 기형율은 대조군에 비하여 현저히 낮았다.

• 한국수정란이식학회지
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• 제6권1호
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• pp.25-32
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• 1991

The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5$\times$106 cells/ml; 71.6%, 5.0$\times$ 106/ml; 71.9%, l.0$\times$ 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5$\times$ 106 cells/mi; 43.6%, 5.0$\times$ 106/ml; 46.8%. l.0$\times$ 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.

• 한국가축번식학회지
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• 제13권3호
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• pp.171-178
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• 1989

These experiments were carried out to investigate the effect of a co-culture with granulosa cells on in vitro maturation, fertilization and development of bovine follicular oocytes. 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 diameter 2-6mm. Bovine oocytes were matured in vitro for 24-26 hr and then fertilized in vitro using epididymal spermatozoa capacitated by preincubation for 2-3hr in BO solution containing BSA(5mg/ml) and caffein(25mM). Eight hours after insemination, the oocytes were cultured in a co-culture system with granulosa cells. The rates of maturation of the follicular oocytes cultured in a co-culture system with granulosa cells were 83.1%, the rate of fertilization of the follicular oocytes culture in a co-culture in a co-culture system with granulosa cells were 76.9%, respectively. No significant difference are observed between control and treatment in maturation and fertilization rates. The rates of embryos developed to 2-, 4-, 8-, 16-cell and monula stages after co-cultured with granulosa cells were 65.8, 57.9, 39.5, 34.2 and 34.2%, respectively. The value for 16-and morula stages were significantly higher (P<0.05) than those of the embryos cultured in the basic medium.

• 한국가축번식학회지
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• 제14권1호
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• pp.1-8
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• 1990

These experiments were carried out to investigate the effects of cumulus cells for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6 mm of diameter. Bovine oocytes were matured in vitro for 24~26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hours in BO solution containing BSA(5mg/ml) and caffeine(2.5mM). Insemination was made by introducing about 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and HEPES(25mM), cultured for 7~8 days with 10~15 eggs/well in 4-well multidishes(Nunc Co.) forming cumulus cell monolayer. The results obtained in these experiments were summarized as follows ; 1. The majority of the follicular oocytes with compacted cumulus cells existed in GV stage while those with dispersed or denuded cumulus cells existed GVBD and M II stage. 2. After 24~26 hours maturation, the maturation rates of the follicular oocytes cultured in TCM-199 containing hormones were slightly higher than those of oocytes cultured in medium without hormones, and the frequency of cumulus compacted or denuded oocytes reaching M II stage cultured in medium containing hormones was 75.7% or 51.7%, respectively(P<0.05). 3. After 20 hours in vitro insemination, percentages of ova fertilized were 61.4% or 51.4%, respectively, for cumulus oophorus intacted or removed, and increased frequency of ova with both male and female pronuclei was found when cumuli were present(P<0.05). 4. The rates of embryos developed to 2-, 4-, 8-, 16-cell and morula or blastocyst stage after cocultured with cumulus cells were 65.0%, 45.3%, 34.7%, 28.0% and 22.7%, respectively. The results for momla or blastocyst stage were significantly higher than those of the embryos cultured in the basic medium(P<0.05).

• Reproductive and Developmental Biology
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• 제39권4호
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• pp.117-125
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• 2015

Iron is required for cell viability but is toxic in excess. While the iron-mediated malfunction of testicular cells is well appreciated, the underlying mechanism(s) of this effect and its relationship with fertility are poorly understood. Ferritin is a ubiquitous intracellular protein that controls iron storage, ferroxidase activity, immune response, and stress response in cells. Ferritin light chain protein (FTL) is the light subunit of the Ferritin. Previously, we had identified the FTL in bovine spermatozoa following capacitation. In present study, to investigate the role of Ferritin in sperm function, mice spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of sodium nitroprusside (SNP), an iron donor. SNP was increased Ferritin levels in a dose-dependent manner. The Ferritin was detected on the acrosome in spermatozoa by immunocytochemistry. Short-term exposure of spermatozoa to SNP increased tyrosine phosphorylation and the acrosome reaction (AR). Finally, SNP affected a significant decrease in the rate of fertilization as well as blastocyst formation during early embryonic development. On the basis of these results, we propose that the effects of Ferritin on the AR may reduce overall sperm function leads to poor fertility in males and compromised embryonic development.

• 한국동물생명공학회지
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• 제35권1호
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1997년도 제34차 추계 학술대회
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• pp.81.2-82
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• 1997