• Journal of Embryo Transfer
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• v.15 no.1
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• pp.39-46
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• 2000

This study was conducted to investigate the effect of hormones, protein sources and anti-oxidants on in vitro maturation (IVM) and in vitro fertilization(IVF) of bovine follicular oocytes. The rates of Holstein follicular oocytes classified as grade A and B(50.2% and 33.2%) were higher than those of Hanwoo cattle(40.3% and 32.0%, P<0.05). The cumulus cell expansion rates of oocytes cultured in TCM-199 and Ham's F-10 medium supplemented with 10% FCS and hormones were higher (81.9~87.6%) than those of non-treated groups (74.5~81.7%). The fertilization rates of oocytes cultured in TCM-199 and Ham's F-10 medim supplemented with 10% FCS, 1% BSA and 10% bFF was 53.8~55.0%, 51.4~52.6%, and 47.0~50.0%, respectively. The polyspermy rates was 13.6~14.2%, 10.0~11.1%, and 10.0%, respectively. When the oocytes were cultured in TCM-199 and Ham's F-10 medium with 50${\mu}{\textrm}{m}$ $\alpha$-tocopherol, the fertilization rates was 62.0 and 60.2%, respectively. In the maturation medium added of 100${\mu}{\textrm}{m}$ cysteamine, the fertilization rates was 64.7 and 66.7%, respectively. The fertilization and polyspermy rates of treated groups were higher than those of non-treated group. The results show that hormones, protein sources and anti-oxidants can provide a benefit for in vitro maturation and fertilization of bovine follicular oocytes.

• Korean Journal of Food Science and Technology
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• v.20 no.6
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• pp.762-768
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• 1988

The lipolytic enzyme of milk from hormone treated and non treated cows was isolated and purified, It was shown that the crude lipase extract from the milk before and after a hormone treatment of the cows was different in color, foaming properties, yield and specific activity. Final purification of the lipase system was achieved by affinity chromatography on Heparin-Sepharose CL-6B. The lipase bound by Heparin-Sepharose was then characterised. The pH-optimum of the purified enzyme was 8.5 for butteroil emulsion as a substrate and the optimum temperature was $30^{\circ}C$ respectively. The molecular weight. determined by SDS-polyacrylamidegel electrophoresis, was about 70,000. The activity increased by 10% hen 0.01% bovine serum albumin was added to the substrate. The results indicate the enzymes obtained by affinity chromatography from milk before and after hormone treatment had the similar characteristics. The second lipolytic active component that was not bound by Heparin-Sepharose must be the cause of spontaneous rancidity.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.1-7
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• 1995

This study was accomplished to illuminate factors of contamination of microbes in the culture medium and effect of antibiotics on prevention of contamination in the medium when bovine follicular oocyte was matured, fertilized and developed in vitro. 1. When washed or unwashed semen diluted with TCM 199 was incubated for 24∼72hr, contamination was come out. 2. When diluted semen with TCM 199 which has penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out only in kanamycin. 3. When imported semen which was diluted in TCM 199 with penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out in all treatments. 4. When semen which was diluted in BO, CZB, Ham's F10 or TCM 199 was incubated for 24∼72hr, kanamycin showed no contamination in all treatments, but gentamycin showed contamination in CZB, Ham's F10 and TCM 199. 5. When the semen diluted in BO was moved at 24hr after incubation into BO and incubated for 72hr, contamination was not come out, but when it was moved into the TCM 199 and incubated for 72hr, contamination was come out at 48 to 72hr of incubation. 6. When the semen diluted in BO, BO＋BSA or BO＋FBS containing gentamycin, kanamycin or nystatin was incubated for 24∼72hr, the diluted semen in BO or BO＋BSA showed no contamination in all antibiotics but the diluted semen in BO＋FBS showed no contamination only in kanamycin. 7.The Pseudomonas cepacia, Serratia liquefaciens, Klebsiella pneumaniae was respectively isolated in the semen of A, B, and C bull and the microbes are highly affected by amikacin, tobramycin and kanamycin. 8. When bovine folicular oocyte was in vitro matured, fertilized and developed in the simple medium with kanamycin, 26.6% was developed to over 32cell stage embryo.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.759-764
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• 2001

This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $38.5^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. The 1~6 passages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for $15{\mu}s$ was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with $10{\mu}M$ calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in $500{\mu}l$ of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at $38.5^{\circ}C$. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1~6 passages were 37.5~44.4%. However, there were no significant differences among passages. These results indicate that 1~6 passage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.227-234
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• 1995

This study was carried out to investigate the effects according to the time course of glucose exposure on the development of one-cell embryos beyond morula in CR$_laa$ medium. One-cell zygotes from B6CBA F$_1$ mice were recovered at 24 ~ 25h after hCG and cumulus cells were removed with 0.1% hyaluronidase. The embryos were pooled and subsequently divided into each groups and cultured in CR$_laa$ at 37$^{\circ}C$ in 5% CO$_2$ in aIr. The embryos were either, placed in CR$_laa$ containing various concentration (5.5, 16.5, 27.5 and 38.5 mM) of glucose for 1 min. and subsequently returned to the fresh culture medium (without glucose), or were transferred to the same media containing glucose at 72 h post hCG. The results obtained in these experiments were summarized as follows: 1. The development rates of zygotes, recovered from the oviducts in M2 and cultured in CR$_laa$ with 3mg/Im FAF-BSA, to expanded blastocysts (25.7%) and hatching bIastocysts (17.6%) were significantly higher than those of zygotes recovered in TL Hepes (0% and 0%, respectively). 2. The development rates of one-cell embryos exposed to 27.5 mM glucose at 72 h post hCG for 1 min, were 68.8% (CR$_1$+BSA) a and 77.1% (CR$_1$+FBS) of expanded blastocyst stage, but there were no significant differences between the embryos exposed for 1 min. or transferred at 72 h. 3. Regardless of glucose concentration (5.5, 16.5, 27.5 & 38.5mM), 45.7~61.5% of embryos developed to the blastocyst stage. There were no significant differences between any of the treatments on the devel-opment of one-cell embryos. Therefore, the detrimental effect of highly concentration was not appeared.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.1
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• pp.7-12
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• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• Korean Journal of Food Science and Technology
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• v.34 no.4
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• pp.732-736
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• 2002

The immunostimulating activities of the hot-water extract from Codonopsis lanceolata were investigated. The proliferation of BSA-primed lymph node cells was enhanced between 2.8- to 11.2-fold compare to control, when cultured with 1 to $25\;{\mu}g/mL$ of C. lanceolata extract. It showed strong immunopotentiating activity than ginseng extract and as remarkable as Bifidobacterium adolescentis M101-4 known as a positive immunostimulator. The proliferation of splenocytes and Peyer's patch cells was enhanced between 4.2- to 13.8-fold and 3.1- to 6.9-fold, respectively, when cultured with 1 to 25 $25\;{\mu}g/mL$ of C. lanceolata extract. It enhanced the production of cytokines such as $TNF-{\alpha}$ and IL-6 in the culture of RAW 264.7 macrophage cells. In the culture of lipopolysaccharide-stimulated RAW 264.7 cells, production of cytokines was as compared to controls. In unstimulated RAW 264.7 cells, both $TNF-{\alpha}$ and IL-6 production were enhanced between 12.6- to 67.8-fold and 2.8- to 10.1-fold, respectively. The hot-water extract from C. lanceolata is expected to be a safe immunopotentiator to maintain the host immunity and develop a physiologically functional food.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.62-62
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• 2003

This study was carried out to investigate the effects of activation agents on parthenogenetic activation of pig oocytes matured in vitro. The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin $B_{l2}$, 25 mM Hepes, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. 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. Cumulus-free oocytes involving first polar body were activated by exposure to various concentrations of ethanol and exposure time of ethanol in Hepes-buffered NCSU23 medium. Also, oocytes were activated by electric pulse alone or combination with ethanol. For electrical activation, oocytes were rinsed twice in 0.3 M mannitol solution supplemented with 0.1 mM CaC1$_2$, 0.2 mM MgC1$_2$, 0.5 mM Hopes and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm apart which was overlaid with the same activation solution. Oocytes were activated with a single DC pulse of 1.3 ㎸/cm for 30 $\mu$sec. After activation treatments, oocytes were washed three times with Hepes-buffered NCSU23 medium and were washed twice with NCSU23 culture medium containing 0.4% BSA, and then cultured in 500 ${mu}ell$ of the same medium for 20 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly more oocytes (29.3~33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8 to 15 min. Electric pulse treatment followed by exposure to ethanol significantly improved the rate of oocyte activation (61.9%) compared with that of other 3 treatments. In conclusion, the optimal activation treatment of ethanol exposure alone for the in-vitro matured pig oocytes was 8% ethanol for 8 to 15 min. Electric pulse treatment followed by ethanol exposure significantly improved the rate of activation.n.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.