• Asian-Australasian Journal of Animal Sciences
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• 제8권6호
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• 한국가축번식학회지
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• 제21권2호
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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.

• 한국가축번식학회지
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• 제13권1호
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• pp.32-39
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• 1989

The present studies were conducted to investigate the possibility of capacitation of bull spermatozoa in the uteri isolated from estrous hamsters and to investigate the possibility of in vitro fertilization of follicular oocytes matured in culture, using ejaculat spermatozoa preincubated in the uteri isolated from estrous hamsters. The follicuar oocytes matured in culture were not fertilized after insemination spermatozoa preincubated for 3 and 5.5-7 h in the uteri isolated from estrou hamsters, but 5, 77, 85 and 50-67% of those oocytes were fertilized by spermatozoa preincubated for 3.5, 4, 4.5 and 5h in those uteri, respectively.

• 한국가축번식학회지
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• 제14권2호
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• pp.93-100
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• 1990

These experiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro maturation of bovine follicular oocytes. Antisera to bovine cumulus cell were produced Japanese Ginat rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified by ammonium sulfate precipitation and Sepharose CL-4B protein-A affinity chromatography. The bovine cumulus cell-specific antibodies were confirmed by indirect ELISA. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coated plates was very high in both intact and solubilized cumulus cells. Namely, the optical density at 1:12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. 2. When the follicular oocytes were treated with antibody to intact cumulus cells, the maturation rate of cumulus compacted and removed oocytes was ranged 47.6 to 59.1%. These value is significantly lower(p<0.05) than that(78.8%) of follicular oocytes cultured without the antibody. 3. the maturation rate of cumulus compacted and removed oocytes treated with antibody to solubilized cumulus cells was ranged 46.7 to 59.1%, significantly lower(p<0.05) than that(82.1%) of ooyctes cultured in antibody free medium. From above mentioned results, it could be said that cumulus cells promote nuclear maturation of follicular oocytes and that the beneficial effect of cumulus cells to the oocyte maturation is inhibited by the action of antibody to cumulus cells.

• 한국수정란이식학회지
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• 제13권2호
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• pp.107-115
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• 1998

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.0$\pm$11.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.7$\pm$1.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

• 한국수정란이식학회지
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• 제9권3호
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• pp.261-268
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• 1994

Immatured bovine follicular oocytes added with serum, hormones, granulosa cells and bovine oviduct epithelium cells were fertilized in vitro after in vitro maturation. In vitro maturation and early development capacity were examined and IVF-derived embryos were transferred and to recipients and effects of sperm treatment on in vitro capacitation were investigated. The rate of in vitro maturation was improved when they were co-culutred with granulosa cells in the TCM199 medium added with 10% FCS and hormones. The percentage of acrosome reaction was not differed between sperm treatments and sperm of above 25% under-went AR during 30 min preincubation with caffeine and heparin. The cleavage rate of oocytes in vitro fertilized in TCM199 medium added with 10% FCS and hormones, GC or BOEG higher than that in medium with 10% FCS and GC. But the rate was not significantly different between GC and BOEG The cleavage of rate oocytes cultured in medium containing serum, hormones and BOEG was 80.2% and more embryos were developed to Blastocyst (17.3%). The selected embryos were transferred to 9 recipients by surgical or nonsurgical method but did not result in pregnancy.

• 한국가축번식학회지
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• 제18권2호
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• pp.121-126
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• 1994

This study was conducted to examine the condition of activation of the nuclear transplant bovine embryos. In vitro fertilized(IVF) and nuclear transplant embryos(NTs) were co-cultured with bovine oviduct epithelial tissue(BOET). NTs were treated with cycloheximide(CHXM) for 0 to 6 h after electrofusion to investigate the activation conditin of recipient ooplast. Then, the infljence of the CHXM treatment timing on the cleavage and development of NTs were investigated in relation to the nuclear transplant time. The cleavage rates of NTs were increased with the increasing time of the CHXM treatment from 0 to 6 h (54.7 to 91.3%, P<0.01). Similar trend was shown in the development into the morula or blastocyst stage, but very limitted. Activation of enucleated oocytes prior to fusion enhanced development of NTs compared with that post fustion. This result suggests that the frequency of activation of NTs can be greatly enhanced by treating with CHXM for 6 h. The result also suggests that if blastomeres of unknown cell cycle stage are used, activation of enucleated oocytes prior to fusion enhances development of NTs.

• 한국수정란이식학회지
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• 제6권1호
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• pp.25-32
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• 1991

The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5$\times$106 cells/ml; 71.6%, 5.0$\times$ 106/ml; 71.9%, l.0$\times$ 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5$\times$ 106 cells/mi; 43.6%, 5.0$\times$ 106/ml; 46.8%. l.0$\times$ 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.

• Asian-Australasian Journal of Animal Sciences
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• 제14권10호
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• pp.1353-1359
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• 2001

The present study was undertaken to assess the influence of dimethyl-sulfoxide plus sucrose solution as a cryoprotectant on the adenosine triphosphate (ATP) content, the ultrastructure and the embryonic development of bovine oocytes matured in vitro. We measured the amount of ATP in cumulus cells enclosed oocytes (CO) or denuded oocytes (DO) equilibrated with or removed from the cryoprotectant (1.5 M DMSO + 0.25 M sucrose + 20% fetal bovine serum in physiological saline). As a result, the ATP contents in both CO and DO, equilibrated with the cryoprotectant, were significantly lower (p<0.05) than that of the each control group. However, ATP content of DO was recovered to the level of the control group ailer removal of the cryoprotectant, but failed to restore for CO. In the observation of the ultrastructure by a transmission electron microscope, all of the mitochondria in the ooplasm of CO and DO equilibrated with the cryoprotectant were swollen with peripherally located cristae following decreased electron density. However, a large proportion of these swollen mitochondria were restored to the normal shape which can be observed usually in the control group after removal from the cryoprotectant. To the contrary, the morphology of many mitochondria of the cumulus cells in CO were not recovered to that of the control group after removal of the cryoprotectant. CO with removed cryoprotectant had significantly lower embryonic development up to the blastocysts stage (p<0.05) after in vitro fertilization compared with that in the control group. These results suggest that the addition and removal of a cryoprotectant has a negative effect for the ATP content of cumulus enclosed oocytes. One of the factor(s) causing the lower embryonic development after removal of cryoprotectant, may be associated with ATP metabolism.

• 한국가축번식학회지
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• 제20권3호
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• pp.353-360
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• 1996

The study was conducted to determine the optimal cumulus cells, glucose and superoxide dimutase(SOD) levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for morulae and blastocyst development. Oocytes were cultured for 0~8 days in TCM-199 medium supplemented with 20% FCS, cumulus cells and with different glucose and SOD levels. The results are summarized as follows; 1. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing cumulus cells and 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 21.1, 25.0, 23.3, 17.9 and 26.3, 25.7, 23.1, 19.4% respectively and there was significant differences on the development to the molurae and blastocysts stage among the cumulus cells and glucose levels. 2. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 11.3~24.5% and 17.3~25.0%, respectively. 3. The in vitro developmental rates bovine oocytes cultured in TCM-199 medium containing 100, 200, 300, 500 $\mu\textrm{g}$/ml SOD levels 0~3 and 0~8 days after insemination were 12.5~22.9% and 12.9~22.2%, respectively. Hight levels of SOD(500$\mu\textrm{g}$/ml) significantly reduced the rates ofmolurae and blastocysts stage(P<0.05).