• Journal of Embryo Transfer
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• v.14 no.1
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• pp.23-29
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• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.111-116
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• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• Development and Reproduction
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• v.20 no.2
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.217-222
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• 2008

The techniques of IVM, IVF and IVC of canine oocytes may provide useful information for gamete salvage programs and the conservation of endangered canidae. This investigation has been made to determine the efficiency of in vitro maturation (IVM) as a basic experiment to study the development of canine oocytes after in vitro fertilization (IVF). The rate of oocytes developing to the Mil stage was higher in the hormone treated group (10 IU/ml hCG+eCG, 14.7%, p<0.05) than in the control group (0 IU/ml hCG+eCG, 10.0%). The monospermy and pronuclear rates of canine oocytes were investigated after caffeine treatment on IVF. Canine oocytes were fertilized in the Fert-TALP medium supplemented with 0, 10, 20 or 30 mM caffeine (Fert I, Fert II, Fert III or Fert IV, respectively). The highest pronuclear formation rate was obtained in the Fert I for 24 h IVF (6.7%, 6/89). Therefore, it is believed that unlike in other mammals, caffeine in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.189-194
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• 1997

The ovaries of Korean native cows or heifers were obtained from a slaughter house and kept on 28~3O˚C and transported to laboratory within 2 hrs. The follicular oocytes were collected follicles. The oocytes were matured in vitro for 24 hrs. In TCM-199 supplemented with 35 $\pi$g /ml FSH, 10 $\pi$g /ml LH, 1 $\pi$g /ml estradiol-17 and granulosa cells at 39˚C under 5% $CO_2$ in air. The caudal epididymis of Korean native bulls were obtained from a slaughter house and transported to laboratory within 30 minutes. Swim-up of collected spermatozoa and freezing sperm was layered under 2ml fertilization B. 0. medium in two tissue culture tubes and held at a 45˚C angle for 0~2 hrs. They wrer fertilized in vitro by freezing sperm treated with heparin for 24 hrs, and then the zygotes were co-cultured in vitro with bovine oviductal epithelial cells for 7 to 9 days. The follicular oocytes recovered were classified into 41.7% as grade I, 51.5% as grade II and 6.8% as graed III. The number of oocytes recovered per ovary was averaged 8.3 and they were classifed into 2.3 as grade I, 2.5 as grade II and 2.3 as grade III. The cleavage rate of matured oocytes was significantly(P

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.209-215
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• 1992

These studies were carried out to overcome 2-cell block and in vitro development to blastocysts in vitro fertilization of mouse embryos. The unfertilized ova were obtained by superovulation in ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and the pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryos after 26hrs of incubation with preincubated sperm were evaluatated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The optimal ranges of pH and osmolality of culture media and of sperm preincubation time for in vitro development of in vitro fertilized ova to blastocyst were pH 7.1 to 7.3, 250 to 350 mosmol and 60 to 180 min, respectively. 2. With the media of pH 7.1, 310 mOsm and sperm preincubation period of 120min in another experiment of large sample size, the in vitro fertilized ova was found 66.5% and the in vitro development of in vitro fertilized ova to blastocyst was found 35.8%. From the above results it was concluded that the optimal conditions of pH and osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOam and 120min, respectively.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.170-170
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• 2004

First of all, in vitro production (IVP) of porcine embryos is an important as initial step to improve bio-technical applications such as transgenesis and cloning for xenotransplantation. In recent years, considerable progress has been achieved in the IVP embryos using advanced methods for in vitro maturation (IVM) and fertilization (IVF). (omitted)

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.117-121
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• 2002

This study was carried out to investigate the in vitro fertilization rate of canine immature oocytes cryopreserved by vitrification freezing. The vitrification solutions of EPS and EDS were consisted of 40％ ethylene glycol 18％ Ficoll and 0.3M sucrose, and 20％ ethylene glycol, 16.5％ DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10％ FCS, respectively. The oocytes were exposed The developmental rate of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 3.8％, 10.7％, 46％, respectively. to EFS or EDS at $25^{\circ}C$ and loaded into straw fer 30 sec. The straws was slowly immersed into L$N_2$. Fertilization and survival rate was defined as development rate on in vitro culture or FDA-test. 1. The fertilization rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was very low(5.3％～31.4％) than the unfrozen oocyte(60.0％). And the fertilization rate after vitrification freezing of immature oocytes was very higher than that of mature oocytes. 2. The survival rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was 55.0％, 40.0％, 28.6％ and 17.1％, respectively. And the survival rate after vitrification freezing of immature oocytes was slightly higher than that of mature oocytes.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.287-296
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• 2011

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.