• Korean Journal of Poultry Science
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• v.42 no.2
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• pp.109-116
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• 2015

Ogye rooster sperm chromatin status can be detected using well established sperm assays. In this paper, a simple and fast method to monitor rooster sperm chromatin status could be employed in field for assessment of chicken sperm quality. Using standard bright field microscope, Diff-Quik stains can be reproducibly, easily and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of three tested Ogye spermatozoa were 93.53%, 82.42% and 90.63% and normal chromatin rates were 87.96%, 74.25% and 85.10% respectively. However, after freezing, the rates of viability of thawed semen were reduced to 69.58%, 61.98% and 72.20% and normal chromatin rate also reduced to 58.91%, 48.49% and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at $37^{\circ}C$ for 5min, the rates of viability, chromatin normality and sperm head activity were shown as $90.63{\pm}1.28%$, $82.44{\pm}8.09%$ and $66.68{\pm}10.29%$ in fresh semen. However, the rates of thawed semen were reduced to $67.92{\pm}7.55%$, $56.92{\pm}12.15%$ and 47.32{\pm}5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining that could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.251-258
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• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.43-50
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• 2003

The objective of this study was to investigate the effect of cryopreservation by slow and rapid freezing on the sperm motility index, viability and morphology of post-thaw human spermatozoa. After rapid freezing and thawing, sperm motility index was significantly higher (MOT:47.40$\pm$20.06%, VCL : 38.12$\pm$15.58 $\mu$m/s, VSL : 28.19$\pm$14.10 $\mu$m/s, VAP:33.64$\pm$15.15 $\mu$m/s, and HYP 2.77$\pm$2.71%) than slow freezing and thawing(MOT : 43.39$\pm$ 18.79%, VCL .33.91 $\pm$ 13.50 Um/s, VSL . 19.98$\pm$0.88 $\mu$m/s, VAP : 24.60$\pm$11.72 $\mu$m/s, and HYP . 1.33$\pm$1.57% ; P<0.05). But sperm Linearity(LIN) was significantly lower(28.83 $\pm$ 10.35) comparing to the slow freezing method(34.64 $\pm$ 11.36 ; P<0.05). On the other hand, significant difference were not observed MAD, WOB, DNC and DNM by slow and rapid frozen-thawed methods. After rapid freezing and thawing, sperm viability was lower(60 $\pm$ 2.2%) than slow freezing method(62 $\pm$2.1%) and sperm morphology was higher(46$\pm$7.7%) than that(44: 8.3). But there was no significantly These results indicate that rapid freezing method was positive effect of sperm cryopreservation in human.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.121-129
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• 1995

To examine the critical effect of oxygen concentration on embryonic development, in vitro fertilized embryos were cultured in media(TCM199 vs. SOF) supplemented sera(1O% FCS vs. 10% HS) with and without bovine oviduct epithelial cells under two gas atmosphere (5% $CO_2$ in air vs. 5% $CO_2$, 5% $O_2$, 90% $N_2$). Oocytes, obtained from abattoir ovaries, were matured in EGF containing TCM199 medium co-cultured with BOEC for 24 hours, followed by exposure to frozen-thawed, heparin4reated spermatozoa in TALP for 30 hours. And then early embryos(1~2 cell) were cultured in both TCM199 and SOF supplemented with 10% FCS or 10% RS under 5% $CO_2$ in air or 5% COi, 5% $O_2$, 90% $N_2$. Development to morulae and blastocysts was recorded on days 7, after the start of in vitro fertilization. The developmental rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC under 5% $CO_2$, 5% $O_2$, 90% $N_2$(24.4%) were significantly(p<0.05) higher than cultured in SOF with BOEC under 5% $CO_2$ in air(14.1%) at seven days after in vitro fertilization. When early bovine embryos were cultured in TCM 199 and SOF under two different gas atmosphere, there were no significant differences in the developmental rates to morulae and blastocysts between supplements of 10% FCS and 10% HS. The rates of development to morulae and blastocysts were significantly(p<0.01) higher in TCM 199 with BOEC(24.7%) than TCM199 without BOEC(10.9%) under 5% $CO_2$ in air, otherwise SOF without BOEC(36.4%) were significantly (p<0.05) higher than in SOF with BOEC (24.4%) under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In summary, these experiments have proved that the culture system in SOF supplemented 10% ES is effective on in vitro development of early bovine embryos under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In addition, it is effective to development of bovine embryos that TCM 199 should be co-cultured with BOEC and SOF should be cultured without somatic cells under two different gas atmosphere.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.4
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• pp.490-496
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• 2020

A styrofoam box is used as a simple and easy freezing method to preserve animal semen as a livestock genetic source. This study optimized the methods of freezing chikso brindled cattle semen. To test the freezing box, the motility of spermatozoa was compared between two box sizes (length×width×heigh) with the dimensions of 23.5×30.5×22.5 cm and 25.5×46.5×26.5 cm. The motility of thawed sperm from brindled Korean bulls was used to confirm the efficiency of the freezing boxes. The box having a larger inner space with larger horizontal and height measurements supported better motility after thawing (60.4±5.3% vs 67.2±3.1%) with 10 min of exposure time in liquid nitrogen vapor. The optimized freezing space is estimated to be an essential element for successful freezing results and the larger box could be used for production of more than 60 frozen semen straws. These properties are also helpful to optimize the cryopreservation techniques that would control the quality and quantity of semen straws according to different animal species.

• Korean Journal of Animal Reproduction
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• v.3 no.1
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• pp.30-35
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• 1979

A, B and C dilutors were used to make Ka (A plus B (1 : 1)) and Na (B plus C(1 : 1)) dilutors in this experiment. Three aliqots of semen were respectivly diluted 1 : 1 and 1 : 2 (semen: dilutor) with Ka, Na and C dilutors and stored at 5$^{\circ}C$ for 7 days in order to study their livability during storage. Fertility was checked for the diluted semen with Ka, Na and C dilutors. Whole semen and extended semen with Na dilutos with and without DMSO were cold shocked at various temperatures for 10 min. Effects of different 1st and 2nd dilution with A, B, C and Na dilutors and of vessels on freezability of spermatozoa were investigtigated. 1. Extended semen 1 : 2 with Na and C dilutors showed highest live sperm index during storage for 7 days at 5$^{\circ}C$. 2. The components of Na dilutor per 100$m\ell$ were skim milk 2.5g, trisaminomethane 0.54g, citric acid 0.265g, glucose 2.835g, fructose 1.5g, sodium lauryl sulfate, 0.08g, penicillin 0.06g, streptomycin 0.075g, and egg yolk 10$m\ell$. 3. Fertility of diluted semen was higher than that of whole semen. Ka dilutor showed higher fertility than Na and C dilutors, and there was no difference in the fertility between Na and C dilutors. 4. Na dilutor with DMSO showed slightly higher livability than Na dilutor without DMSO during storage for 7 days at 5$^{\circ}C$. 5. Cold shock at 1$0^{\circ}C$ for 10 min. decreased greatly the sperm livalility of whole semen but not of extended semen with Na dilutor. Addition of DMSO to Na dilutor has no effect in prevention of cold shock. 6. The extended semen with C. C dilutor (1st and 2nd dilution with C and C dilutor) showed higher post-thawing sperm livability than A.A and Na. B dilutors. Na. B dilution shwed higher post-thawing sperm livability than A.A dilution. There was no difference in the post-thawing livability between semen in 1$m\ell$ straw and 10$m\ell$ aluminium package.

• 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 Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Development and Reproduction
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• v.10 no.4
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• pp.239-245
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• 2006

Experiments were conducted to determine the effects of beta-mercaptoethanol(${\beta}-ME$) supplements to the maturation medium on in vitro fertilization(IVF) and intracellular glutathione(GSH) concentration. Bovine cumulus-intact oocytes were matured in TCM-199 medium containing FBS, hormonal supplements, and ${\beta}-ME$(0, 25 and $50\;{\mu}M$) for 12h and 24 h. After culture, cumulus-free matured oocytes were co-incubated with frozen-thawed spermatozoa for 24h. Maturation rate increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant differences among treatment groups. Also, increases(p<0.05) in intracellular GSH concentration before and after fertilization were observed in $50\;{\mu}M\;{\beta}-ME$ supplements to the maturation medium. Male pronuclear formations after IVF was increased(p<0.05) in ${\beta}-ME$ treatment group, but no significant difference among treatment groups. In conclusion, supplementing ${\beta}-ME$ into the maturation medium increased maturation rates, fertilization rates, and intracellular GSH concentrations.

• Korean Journal of Poultry Science
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• v.39 no.4
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• pp.291-295
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• 2012

The use of methylacetamide (MA) as a cryoprotective agent for freezing Korean Native Black rooster Ogye semen was examined with artificial insemination. The diluted Ogye semen with HS-1 was subjected for 2 step dilution method of cryopreservation in which the final concentration of MA was adjusted to 7.5%. The sperm viability after thawing was reduced from $95.17{\pm}0.93%$ to $55.93{\pm}1.38%$ which was confirmed by live-death analysis based on Fluorescence-Activated Cell Sorting (FACS). The rates of fertilized eggs with fresh or frozen-thawed semen were reduced from $94.98{\pm}3.93%$ to $66.36{\pm}8.43%$ at day 7 with significant difference. However, the hatching rates of experiments at day 21 did not shown difference between $92.64{\pm}2.33%$ and $90.45{\pm}8.05%$ (P<0.05). With these results, the utilization of MA for freezing of Ogye spermatozoa could affect on viability of frozen-thawed semen but not on the fertility of lain eggs and hatchability of fertilized eggs and also provide possible tools of freezing for poultry genetic resource conservation.