• Journal of Embryo Transfer
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• v.22 no.4
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• pp.219-222
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• 2007

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% $CO_2$, 95% air, $38^{\circ}C$. 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between $48{\sim}70$ hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were $11.5{\pm}1.2%,\;10.8{\pm}1.0%,\;9.6{\pm}1.2%\;and\;12.4{\pm}1.5%,\;11.8{\pm}1.5%,\;11.2{\pm}1.4%$ respectively. This was higher than that in both single and combined stimulated groups compared to control group ($6.2{\sim}7.2%$).

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.135-141
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• 2006

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

• The Korean Journal of Zoology
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• v.31 no.2
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• pp.87-94
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• 1988

Korean frogs (R. dybowskii and R. nigromaculara) were collected from chonnam area and their oocyte maturation was induced by using in ultro follicle culture system. Follicles were isolated from the frog ovary and cultured for 24 hr in (amphibian Ringer's soluion AR) at 22 C in the presence or absence of hormones. Follicular cocytes of R. dybowskii were induced to mature (germinal vesicle breakdown, GVBD) by the presence of progesterone, 0.1 $\mu$g/2 ml and that of R. nigromaculata by 1 $\mu$g/2 ml of progesterone. Follicles of the frogs were also responded to (frog pituitary homogenate FPH) in terms of their cocyte maturation. Follicular cocytes of R. dybowskii were induced to mature by FPH at concentration of 0.01 pituitary equivalent/2 ml and that of R.nigromaculata at 0.1 pit equiv./2 ml. The culture time required for the maturation of bath frog follicles was 915 hr. The responsiveness of the follicles of korean frogs to hormones (progesterone or FPH) was nearly the same as that of R. pipiens which are most commonly used amphibians. Particularly, follicular cocytes of R. dybowskii used from February matured spontaneously without stimulation of hormones during in vitro culture. Furthermore, those cocytes were spontaneous- ly ovulted when the ovarian fragments were cultured in a flask.

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.155-161
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• 2000

The morphological changes on nuclear phase of the germinal vesicle of porcine follicular oocytes during in vitro culture were examined. The high rates (75~77%) of the oocytes collected from follicles of 1~2mm or 6~100mm in diameter were at the GV-I to GV-II stages. When oocytes with or without cumulus cells after collection from follicles of 2~6mm in diameter were cultured for 5 h, the rates of oocytes at GV-IV to GV-Ⅵ stages were higher in oocytes with (52%) than in oocytes without (30%) cumulus cells. After 1 h of oocyte culture, there was no differences in the distribution of GV-IV to GV - Ⅵ stages in the media with or without catalase, xanthine and catalase＋xanthine. After 5 h of culture, however, the distribution of GV-IV to GV-Ⅵ stages were 46, 69, 69 and 70% for medium with none, catalase, xanthine and catalase＋xanthine. The highest rate of GVBD was also observed in the medium with catalase＋xanthine (6%). These results indicate that exposure of porcine follicular oocytes to catalase＋xanthine excels maturation to GV stage and enhances oocyte nuclear maturation.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.243-250
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• 2001

The objectives of the present study were to examine the relationship between catalase (0.1 mg/$m\ell$) and xanthine (5 mM) on in vitro maturation of porcine follicular oocytes. At 48 h after maturation, the proportions of oocytes matured to metaphase-II stage were significantly higher (P＜0.05) in the medium with control (72%), catalase (73%) or catalase plus xanthine (70%) than of oocytes cultured with xanthine (54%). On the other hand, oocytes cultured in medium with catalase and/or xanthine for 30 h were not significantly different in maturation rates (6~l4%). At 36, 42 and 48 h after culture, however, the maturation rates were significantly (P＜0.05) higher in medium with (49~70%) that than without (29~50%) catalase regardless of presence of xanthine. When the oocytes were cultured with periods prolonged in medium with and without xanthine, the maturation rates did increase with high proportions at 72 h of culture. No significant differences, however, were observed in maturation rates between groups with and without catalase. On the other hand, degenerated oocytes were increased with culture periods, the proportions was significantly (P＜0.05) lower in medium with (28%) than without (47%) catalase at 120 h of culture. However, there were no significant difffrences between with and without catalase in medium added xanthine. The parthenogenetic oocytes were observed from 72 h after culture in medium with xanthine, but were no significant differences between with and without catalase. From these results, it is indicated that porcine oocytes nay respond to maturation stimulus by 72 h of culture in medium with catalase and xanthine and that parthenogenesis can be obtained with prolonged culture periods.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.289-299
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• 1993

The polypeptide patterns of cellular and follicular components were analysed by SDS-PAGE and two dimensional(2-D)electrophoresis combined with isoelectric focusing (IEF) to establish protein profiles in each of the components in porcine follicles. Oocyte-cumulus complexes were cultured in M16+FCS+Gn at 39 in an atmosphere of 5% CO$_2$, in air for 35 h. At the end of the culture, the zona-free oocyte, ZP alone and cumulus cells were prepared and analysed either on 10% SDS-PAGE for the protein profile at the first dimensional gel or 2-D protein pattern. The amounts of each samples were determined for the visualization with Coomasie brilliant blue (CBB) or silver staining, thus giving useful information for the identification of specific proteins in the components or appropriate amount of samples for proper visualization. Oocyte showed 25 and 114 kd major protein band. Other minor components were additionally visualized with CBB on the same gel after silver staining procedure. Cumulus cells also showed specific proteins which is not present in the oocytes. The number of cumulus cell was proper to give major bands with CBB and additional minor bands with silver staining. To establish the degree of contamination from the remnant of the corona radiata to the ZP, zonae were differently prepared or analysed by SDS-PAGE.The preparation of the ZP in this study did not showed any contamination judged by the protein profile of the components. Also follicular fluid showed its specific protein profile without any significant differences among the different sizes of follicles. The established protein profile of each follicular component should be helpful for the identification and elimination of contaminated components, i. e., antigen preparation or immunological studies. The results also suggest that the preparation of each components in the study was appropriate and can be used for a further sensitive biochemical analysis in mammalian oocytes and early embryos.

• Journal of Veterinary Clinics
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• v.22 no.2
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• pp.157-159
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• 2005

Two-month old male Shih Tzu weighing 2.1 kg was presented with alopecia in black-haired areas of the skin. The hair loss had been present since five weeks of age. There was no history of pruritus on any part of the body. No other symptoms had been recognized. Physical examination found no abnormalities other than hair loss. Skin examination showed marked alopecia of black-haired area of the body. The white areas appeared to have hair growth of normal density and texture. Affected pigmented areas showed no evidence of skin lesions. Skin scraping and fungal culture were negative. Microscopic examination of plucked black hairs showed marked pigment clumping in the remnants of the follicles and were mainly in telogen phase. But white hairs were normal in various stages of hair growth. The diagnosis of black hair follicular dysplasia was made based on the history, alopecia of the pigmented areas, the confinement of abnormalities to dark areas, and the normal unpigmented areas.

• 대한생식의학회:학술대회논문집
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• 2002.11a
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• pp.81.1-81.1
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• 2002

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.43-50
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• 1998

The effect of oxygen tension on embryonic development in co-culture was evaluated from the standpoint of the reduction of dissolved oxygen concentration by the oxygen consumption of feeder cells. Three co-culture systems using bovine oviductal epitherial cells (BOEC), African green monkey kidney cells (Vero cells) or buffalo rat liver cells (BRLC) have been compared in terms of development of bovine embryos derived from oocytes matured and fertilized in vitro. Among the co-cultured embryo, Vero cells su, pp.rted the highest developmental rate (29%) and the other two showed the similar rates. When the co-cultures were incubated in three different oxygen tension such as 5, 10, 20% oxygen atmosphere, embryos co-cultured with Vero cells at 10%-O2 resulted in the highest percentage of development. From the measurement of oxygen consumption of feeder cells, BRLC consumed 1.38　10-10 mg-O2/min/cell which was higher than 0.94　10-10 and 0.26　10-10mg-O2/min/cell for Vero cells and BOEC, respectively. Based on the oxygen consumption data, the phenomena of optimum oxygen tension required in embryo development in vitro has been analyzed, and we suggested that gas phase oxygen concentration, oxygen consumption rate of feeder cells and the number of feeder cells should be considered for the design of optimal co-culture system for effective fertilization of embryos in vitro.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.13-13
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• 2001

This study was undertaken to evaluate the effects of catalase using xanthine (X) - xanthine oxidase (XO) system on in vitro maturation and fertilization in pig. When follicular oocytes were cultured in maturation medium with X and/or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obrained in culture with X-XO system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, hut were not different in medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with, none (P<0.05), XO and X＋XO. On the other hand, when sperm were treated with none, X, XO and X＋XO, lipid peroxidation were higher in medium without that than with catalase. However, the changes in sperm penetration and lipid peroxidation showed opposite patterns. The sperm suspensions were also treated with X and/or XO for assay of sulfhydryl (-SH) group content. Under the above all conditions, sperm-SH group were higher detected In medium with that than without catalase. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were higher than in medium with X, XO and X＋XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO system may be caused stimulating in vitro maturation and fertilization in pig. This work was supported by grant No. 2000-1-22200-001-3 from the Basic Research Program of the Korea Science & Engineering Foundation.