• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.169-174
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• 2007

Despite many efforts to improving canine in vitro maturation(IVM), the efficiency is still low compared to that of other mammalian species. The present study investigated the effects of gonadotropin, epidermal growth factor and cysteine supplementation on in vitro maturation of canine oocytes. Cumulus-oocyte complexes (COCs) were matured in basic medium (TCM-199 containing 10% FBS, 0.11mg/ml sodium pyruvate supplemented with or without $10{\mu}g/ml$ FSH, 10ng/ml EGF and 0.57mM cysteine) for 72 hr at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After culture, oocytes were stained with Hoechst 33342 $(10{\mu}g/ml)$ for 30 min at $4^{\circ}C$, and assessed their nuclear status (GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphasse I, MII: metaphase II, UK: unknown stage). No differences were observed in GV, MI and MII rate except GVBD rate between with and without gonadotropins addition respectively (6.7%, vs. 17.2%). Supplementation of 10ng/ml of EGF in hormones added IVM medium resulted in significantly (p<0.05) higher MII rate (4.54% vs.7.06%). Although there are no significantly difference, total of MI and MII rates were increased by adding cysteine. In conclusion, the present study indicates that supplementation of $10{\mu}g/ml$ LH and FSH, 10ng/ml EGF and 0.57mM cysteine in canine IVM medium show a positive influence on the progression of maturation to MII at 72 hr.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.1-8
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• 1996

The objective of this study was to determine effects of oviductal fluid on the sperm penetration and subsequent in vitro development of porcine oocytes. The addition of oviductal fluid to the fertilization medium decreased sperm pen etration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid. Chlortetracy cline (CTC) fluorescence patterns were used to determine incidence of capacitation and acrosome reaction. The proportion of capacitated a and acrosome free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid. These results suggest that the factor(s) in secretion from the oviduct reduces polyspermic fertilization and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilization me-dium into an intact acrosome.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.123-128
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• 2005

This study examines the effects of kinds and concentrations of cryoprotectants on the survival rate of vitrification-thawed porcine oocytes, together with the effects on survival, in vitro fertilization and development of immature oocytes. 1. The developmental rate of oocytes to MII and diploid stage when the vitrification-thawed of recovered immature oocytes cultured for 0, 15, 30 and 40h were cultured for 0, 15, 30 and 40h were $56.7\%,\;53.3\%,\;63.3\%,\;65.0\%\;and\;23.3\%,\;18.3\%,\;10.0\%,\; 3.3\%$, respectively. The in vitro development to MII stage were lower than the control group $(78.2\%)$, but higher fo. diploid stage $(5.5\%)$. 2. When the vitrification of immature oocytes after being culture for 0, 15, 30 and 40 hours, the survival rate were $34.0\%,\;26.0\%,\;18.0\%\;and\;10.0\%$ respectively. This result was lower than that of the control group $(60.0\%)$. 3. When the fertilization of the vitrified immature oocytes after being culture for 0, 15, 30 and 40 hours, the in vitro fertilization rate were $60.0\%,\;54.0\%,\;48.0\%,\;38.0\%$, and developmental rates were $26.0\%,\;18.0\%,\;8.0\%,\;4.0\%$, respectively. This results were lower than the control group $(78.0\%\;and\;38.0\%)$. 4. When the fertilization of the immature oocytes after being culture for $0\~15$ hours vitrified with EDS and ETS, the fertilization and developmental rates were $50.0\%,\; 22.0\%$ and $46.0\%,\;18.0\%$, respectively. This results were lower than the control group $(74.0\%\;and\;38.0\%)$.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.85-85
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• 2001

본 연구는 돼지 난자의 체외 수정시 sucrose 첨가가 돼지 난자의 다정자 침입율에 미치는 영향을 조사하기 위하여 실시하였다. 돼지 난포란은 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 $\mu\textrm{g}$/$m\ell$ FSH, 0.5 $\mu\textrm{g}$/$m\ell$ LH, 및 10 ng/$m\ell$ EGF가 첨가된 TCM199 배양액에서 42-44 시간 동안 배양하여 체외 성숙을 유도하였으며, 2 mM caffeine과 0.1% BSA가 첨가된 체외수정용 mTBM 배양액에 3% sucorse를 첨가하여 6시간동안 수정을 유도하였다. 수정된 난자의 다정자 침입율을 조사하기 위해 체외수정 후 12, 13 및 14 시간대에 각각 1% orcein으로 염색하여 전핵 형성율을 조사하였다. 돼지 난자의 체외수정시 3% sucorse를 첨가하였을 때, 전핵 형성율을 조사한 결과 대조구에서 미수정, 1, 2, 및 3 PN 이상의 전핵 형성율은 각각 57.6 7.6, 9.7, 및 25%로, 처리구 74.2, 7.6, 13.6, 및 4.5% 에 비교하여 다정자 침입율이 유의하게 높았다(p<0.05). 그리고 3% sucorse를 첨가한 체외수정용 배양액에서 수정을 유도한 후 12, 13, 및 14 시간대별로 나누어 전핵 형성율을 조사한 결과, 수정 후 시간이 경과되면서 다정자 침입율이 증가되는 것을 볼 수 있었다 그러나 대조구 60.7%에 비해서는 각각 32.5, 36.0, 및 33.3%로 현저히 낮은 것을 볼 수 있었다.(Table Omitted). 또한, 3% sucorse가 첨가된 배양액에서 수정된 수정란의 발달율을 조사한 결과, 배반포 생산율은 처리구 21.8%로 대조구 6.9%에 비해 유의하게 높은 것을 확인 하였다. 따라서, 돼지 난자의 체외수정시 3% sucorse첨가는 체외수정란 생산에서 문제가 되고 있는 다정자 침입율을 저하시키고 배발달율을 향상시킴으로서 수정란의 체외 대량생산 효율성은 물론 수정란 이식시 수태율을 증가시킬 수 있을 것으로 사료된다.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.65-71
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• 2017

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at $40.5^{\circ}C$ in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

• Development and Reproduction
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• v.6 no.1
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• pp.17-23
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• 2002

In oocyte retrieval, a vein anesthetic drug is commonly used for induction and maintenance of general anesthesia. Propofol and Thiopental sodium are frequently used for ultrasound-guided transvaginal oocyte retrieval. The present study aimed to assess the effects of Propofol and Thiopental on in vitro fertilization(IVF). Immature porcine oocytes were exposed to various concentrations ot Propofo1 and Thiopental sodium. The rates of oocyte maturation, fertilization and development were observed. The parthenogenetic effects of the anesthetics were also evaluated. The rate of oocyte maturation after exposure to high concentrations of the anesthetics for long time was significantly higher than that of the control. But the rate of fertilization after long-time exposure to the high concentration of the anesthetic drugs was significantly lower than that of the control. The results support that Propofo1 serves like other anesthetics described, as a parthenogenetic activator. Oocytes exposed to Thiopental sodium showed decreased rates of maturation and fertilization. These results suggest that usage of optimum concentration of anesthetic drug is important in increasing the rates of oocyte maturation, fertilization and development in IVF.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was 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$_{12}$ , 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. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.191-195
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• 1995

Bovine follicular oocytes were matured in vitro for 3D hr and exposed to Dulbecco's phosphate buffered saline containing 7% ethanol for 7 minutes for their activation, and incubated in TCM199 containing 10% fetal calf serum (FCS) and cytochalasin D (CD). When the oocytes were exposed to cytochalasin D for 0, 5, 10 and 15 hr, 5 hr showed highest developmental rate to blastocyst (16%), expanded blastocyst (13%) and hatched blastocyst (7%). Also when the oocytes were cultured for 7 hours in medium contai ning 0, 2.5, 5 and 7.5 ${\mu}\textrm{g}$/ml cytochalasin D, 2.5 ${\mu}\textrm{g}$/ml showed highest developmental rate to blastocyst (13%), expanded blastocyst (7%) and hatched blastocyst (4%). In conclusion, cytochalasin D played very important role for parthenogenetic development of follicular oocytes and 5 hr of exposure time to CD and 2.5 ${\mu}\textrm{g}$/ml CD in the medium was optimal for development of the follicular oocyte.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.157-161
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• 2003

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of cats oocytes and development of IVM/IVF embryos. The results were summarized as follows : 1. The fertilization and developmental rate of fresh and salts-stored oocytes with and whithout cumulus cells were 65.7%, 17.1% and 28.6%, 8.6% and 57.1%, 13.3%, 23.3%, 3.3%, respectively. The rate of oocytes with cumulus cells(13.3%∼65.7%) was higher than that of denuded oocytes(3.3%∼28.6%). 2. The fertilization and developmental rate of oocytes recovered from ovaries collected at different stages of the reproductive cycle were 68.9%, 44.4%, 48.9% and 17.8%, 8.9%, 12.8%, respectively. 3. The fertilization and developmental rate of oocytes in vitro cultured at different time of incubation(24, 36 and 48 h) were 66.7%, 46.7%, 48.9% and 17.8%, 11.1%, 8.5%, respectively. respectively. The rate of oocytes incubated 24 h(66.7%) was higher than that oocytes incubated 36 and 48 h(46.7%∼48.9%). 4. The fertilization and developmental rate of oocytes treated activation and non-activation oocytes were 57.4%, 31.4% and 22.9%, 11.4%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.