• Journal of Embryo Transfer
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• v.21 no.1
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• pp.1-5
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• 2006

본 연구는 개 채취 정액의 동결후의 생존성과 신선 및 동결 정자의 capacitation, acrosome reaction과 생존성을 조사하고, 아울러 신선 및 동결 정액을 자연 발정 또는 발정 유기 암캐 에게 인공수정 후 임신율을 조사하였다. 개 채취 정액의 동결 융해 후의 생존성은 $64.5{\pm}2.30%$로서 신선 정액의 생존성에 비해 유의하게 낮게 나타났다(p<0.05). 신선 및 동결 정액의 capacitation, acrosome reaction 및 생존성은 각각 $52.5{\pm}4.5%,\;9.5{\pm}0.6%,\;68.8{\pm}4.5%$ 및 $16.2{\pm}3.2%,\;3.2{\pm}0.5%,\;24.5{\pm}2.5%$로 나타났다. 신선 및 동결 정액을 자연 발정 또는 발정을 유기한 암캐에 인공수정했을 때 임신율은 각각 50.0% 및 33.3%로서 동결 정액을 이용했을 때 임신율이 신선 정액에 비해 낮은 임신율을 나타냈다.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.217-224
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• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• Toxicological Research
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• v.33 no.4
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• pp.315-323
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• 2017

Preconceptual sex selection is still a highly debatable process whereby X- and Y-chromosome-bearing spermatozoa are isolated prior to fertilization of the oocyte. Although various separation techniques are available, none can guarantee 100% accuracy. The aim of this study was to separate X- and Y-chromosome-bearing spermatozoa using methods based on the viability difference between the X- and Y-chromosome-bearing spermatozoa. A total of 18 experimental semen samples were used, written consent was obtained from all donors and results were analysed in a blinded fashion. Spermatozoa were exposed to different pH values (5.5, 6.5, 7.5, 8.5, and 9.5), increased temperatures ($37^{\circ}C$, $41^{\circ}C$, and $45^{\circ}C$) and ROS level ($50{\mu}M$, $750{\mu}M$, and $1,000{\mu}M$). The live and dead cell separation was done through a modified swim-up technique. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. The results indicated successful enrichment of X-chromosome-bearing spermatozoa upon incubation in acidic media, increased temperatures, and elevated $H_2O_2$. This study demonstrated the potential role for exploring the physiological differences between X-and Y-chromosome-bearing spermatozoa in the development of preconceptual gender selection.

• Journal of Life Science
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• v.24 no.11
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• pp.1252-1257
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• 2014

While dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant agent in the cryopreservation of cultured mammalian cells, it has been reported to cause differentiation of some cell lines by DNA methylation and associated histone modifications. To avoid the side effects of DMSO in cryopreservation, other agents might be more appropriate for maintaining the stable differentiation of cultured cell phenotypes through cryopreservation. All cryoprotectants should be highly soluble in water and display low cell toxicity. Cryoprotective agents have been shown to be effective in animal sperm preservation, and eight types of amides were examined in the cryopreservation of cultured mouse endothelial cells. Among the amides examined, acetamide and lactamide were effective cryoprotectants for cultured mammalian cells. The most effective concentration of lactamide, 1.5 M, had an even lower cryoprotective ability than 1M DMSO. Because successful cryopreservation of cultured cells is hampered by osmotic stress, the adequate ionic concentration was determined by diluting phosphate-buffered saline (PBS) in the 1.5M lactamide solution. The most effective concentration was $0.4{\times}PBS$, which minimized osmotic stress during the cryopreservation of cultured cells. As the addition of high molecular weight materials in cryopreservation media improves the viability of cells, the effects of bovine serum albumin (BSA), hydroxyethyl-starch (HES), and dextran were examined. The best combination of lactamide-based media for cryopreservation was found to be 1.5 M lactamide in $0.4{\times}PBS$ with 1% BSA.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.137-141
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• 2011

These study was to investigate the in vitro fertilization and viability of fresh and vitrified oocytes. Also, the developmental capacity of IVF and intracytoplasmic sperm injection (ICSI) oocytes were investigated. Then vitrification was performed with the use of 20% ethylene glycol + 20% DMSO + 0.5 M sucrose + 10% FCS + TCM-199 medium. Vitrification immature oocytes are cultured in vitrification solution for 10 min afterwards transferred to expose at room temperature for 5 min. and transferred to the ice water for 5 min. The oocytes were sealed in a 1.0 mm straw and placed in a $LN_2$ container. Frozen oocytes were rapidly thawed in a water bath at $30{\sim}35^{\circ}C$, and then placed in TCM-199 medium containing 0.5 M sucrose for 5 min each, respectively, at $38^{\circ}C$. After being washed for 2~3 times, using fresh medium the oocytes were cultured in TCM-l99 medium supplemented with 5% FCS at $38^{\circ}C$ in 5% $CO_2$ and air. The normal morphology of fresh and vitrified-thawed oocytes were $87.1{\pm}2.1%$ and $54.8{\pm}2.5%$, respectively. The viability rates of fresh and vitrified-thawed oocytes were $70.0{\pm}2.2%$ and $41.9{\pm}2.6%$, respectively. Viability rates of vitrified-thawed oocytes were lower than that of fresh follicular oocytes (p<0.05). The in vitro maturation rates of fresh and vitrified oocytes were $45.1{\pm}3.6%$ and $28.9{\pm}4.4%$, respectively. The IVF rates of fresh follicular and vitrified-thawed oocytes were 34.00.2% and $20.2{\pm}2.6%$, respectively. The in vitro maturation and fertilization rates of vitrified-thawed oocytes were lower than those of the fresh follicular oocytes (p<0.05). A total of 350 oocytes were fixed and stained after co-incubation with spermatozoa, of which 88 had identifiable nuclear material. After IVF for 20 hrs, $25.1{\pm}3.4%$ of the oocytes found to have been penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, and 105 oocytes contained identifiable nuclear material. After IVF and ICSI for 20 hrs, $34.3{\pm}3.4%$ and $59.0{\pm}2.0%$ of the oocytes were found to have been penetrated by spermatozoas. The developmental rates upon ICSI were significantly higher than those of the IVF method (p<0.05).

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.25-32
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• 2002

This study were performed to investigate the effect of Bisphenol A(BPA) administration on semen characteristic and body or organ weight in mice. In BPA administration for 15 days or 30 days, sperm concentration and viability were lower in group treated with BPA than in control group. The proportion of sperm abnormality were significantly(P＜0.05) increased in groups administrated with BPA for 15 or 30 days than in control group. The administration of BPA in mice didn't affect the body and reproductive organ weight such as testis, epididymis, vasicular gland and coagulatin gland. The spleen weight were significantly affect, but liver and kidney weight were not affect in BPA administration groups for 15 days or 30 days This studies indicate that the short or long term BPA administration in mice were noxious effects on the semen characteristics and organ weight, but didn't affect body weight and reproductive organs.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.189-203
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• 1999

To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.

• Journal of Life Science
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• v.25 no.7
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• pp.765-772
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• 2015

The aim of this study was to determine the effect of decursin (D) and decursinol angelate (DA) against bisphenol A (BPA) toxicity in a rat two-generation study. Adult rats were divided into the following three groups: (1) control, (2) BPA, and (3) BPA+D/DA. The D and DA treatment of F0 parents increased the terminal body weight and relative adult organ weights (testes, kidneys, spleen, and liver) when compared with the BPA group. A significant decrease in sperm count was found in the BPA+D/DA (7.69%) and BPA (64.70%, p<0.01) groups, when compared with the sperm count in the control group. No offspring were obtained in the F1 generation of the BPA (50 mg/kg/day) group, but the addition of D/DA in the BPA+D/DA group significantly restored fertility (55.78%) and gestation indices (98.87%) in the F1 generation. No significant differences were found in the fertility index between the control (75.02%) and the BPA+D/DA (78.11%) groups in the two-generation study, when compared with the one-generation study. The viability ratio during lactation in the D/DA group was also similar to that of the control group. These data indicate that D/DA (50 mg/kg/day) administered over two generations causes significant positive changes in reproductive or developmental parameters.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.90-90
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• 2002

The purpose of this study was to investigate the effect of kind and combination of sugars on the viability and acrosome damage of post-thaw spermatozoa in canine. The extender used was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as single (fructose, xylose, trehalose), two combinations (Fruc+Tre, Fruc+xyl, Tre+xyl) and three combinations (Fruc+Tre+Xyl). The concentration of sperm collected were adjusted of 50${\times}$10$\^$6/ per straw for freezing. The frozen spermatozoa were thawed at 37$^{\circ}C$ for 1 min and then analysis for CASA program in Livestock Improvement Main Center, NACF. The motility of post-thaw spermatozoa in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (79％ vs. 63, 66, 70, 71, 74 and 75%). The progressive motility after CASA analysis in Fuc+Tre group was also higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (67% vs. 53, 57, 60, 61, 62 and 64%). The acrosome damage of post-thaw spermatozoa stained was not significantly different among treatment groups such as fructose, trehalose, xylose, Fru+Tre, Fru+xyl, Tre+xyl and Freu+tre+xyl (17.7, 18.3, 28.0, 17.0, 19.7, 20.0 and 19.0%). The results indicated that the motility and progressive motility of post-thaw spermatozoa in Fru+Tre group was better, and acrosome normality was not different among all groups. The use of Tris-buffer supplemented with Fru+Tre as sugar for frezing of canine spermatosoa could be better and apply to semen banking and artificial insemination.

• Korean Journal of Veterinary Service
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• v.31 no.4
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• pp.547-554
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• 2008

Bacteriospermia is a frequent finding in fresh boar semen and can result in detrimental effects on semen quality and longevity. The objectives of this study was to evaluate types of bacterial contaminants in porcine fresh semen and the reducing effect of antibiotic and density gradient with percoll on the bacterial contaminants. Fresh semen was collected by gloved-hand method into a pre-warmed($37^{\circ}C$) thermostable bottle, and was inoculated onto blood agar and MacConkey agar, respectively. After incubated for 48 hour, 7.5% $CO_2$ at $37^{\circ}C$, bacterial colonies were selected and identified by Gram staining, oxidase test, catalase test and finally identified using API kits and Vitek system. Aerobic culture yielded a variety of bacteria from different genera. The most prevalent contaminant of fresh semen were Leclecia adecarboxylata, Acineobacter banmanni, Staphylococcus epidermidis, Staphylococcus cohni spp urealyticus, Proteus mirabilis. Most of identified bacteria were Gram(-) and non-pathogenic bacteria. It seems that bacterial contaminants in fresh semen were seem originated from multiple sources at the stud/farm, and were from animal and non-animal origins. Gentamicin treatment did not eliminate the bacterial contaminants completely but 3 step-density gradient with percoll completely removed the bacterial contaminants in fresh semen. Therefore, future study is necessary to prove that density gradient method with percoll can eliminate bacteria in fresh semen without significantly affecting sperm viability or function.