• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.181-185
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• 2004

This study was conducted to examine whether activation treatments, source of oocytes and culture conditions affect in vitro developmental ability of caprine oocytes. Mature Korean native goats were pretreated with intravaginal CIDR for 10 days. The goats were then treated with a single intramuscular injection of 1,000 IU PMSG on Day 8 or twice daily injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR insertion for superovulation. All the goats were injected with 10 mg PGF/sub 2a/ on Day 8 and 400 IU hCG on Day 10 of CIDR. Oocytes were surgically collected by oviduct flushing(in vivo maturation) or direct follicle aspiration(in vitro maturation) through mid-ventral incision at 35 h after hCG injection. Fifteen to twenty oocytes were placed in TCM-199 medium containing 25 mM Hepes and hormones under mineral oil at 39℃ in a humudified atmosphere of 5% CO₂ in air for 22 to 24 h. After maturation, the oocytes were activated by electric stimulation or ionomycin + 6-DMAP. The activated oocytes were then cultured in M16, TCM-199 and mSOF media supplemented with proteins at 39℃ for 6 to 7 days. Activation treatments did not affect cleavage of the oocytes. The cleavage rates were 64.1% (41/64) in oocytes activated by electric stimulation and 76.5% (218/285) in oocytes activated by ionomycin + 6-DMAP. The proportion of development to blastocyst was 15.6% (34/218) in oocytes activated by ionomycin + 6-DMAP, but activation by electric stimulation did not support embryos developed beyond morula stage. There were no differences in the cleavage rates of activated oocytes experiencing in vivo (86.8%, 66/76) and in vitro maturation (69.0%, 127/184). However, the development rate to blastocyst stage was significantly (P<0.05) higher for oocytes matured in vivo (50.0%, 33/66) compared to in vitro (0.8%, 1/127). Culture conditions did not affect the cleavage of -activated oocytes. The cleavage rates were 51.6% (49/95) in M16, 64.3% (18/28) in TCM-199 and 81.0% (145/179) in mSOF, respectively. By contrast, the development rate of activated oocytes to stage was greater (P<0.05) for oocytes cultured in mSOF medium (23.4%, 34/145) than in M16 or TCM-199 (0.0%). Our results suggest that source of oocytes and culture conditions are major factors affecting in vitro development of caprine parthenogenetic oocytes.

• Journal of The Korean Society of Grassland and Forage Science
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• v.22 no.4
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• pp.265-272
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• 2002

Forage breeding laboratory of National Livestock Research Institute, R.D.A. has made interspecific hybrids of Lolium multiflorum $\times$ L. pratensis and intergeneric hybrids of Lolium $\times$ Festuca since 1984, and has collected ecotypes of Italian ryegrass since 1991. Growth characteristics of these hybrids and ecotypes were researched, and then these clone lines were named. Among these clone lines, the several clones that have polen fertility, high cold-tolerance, and similar heading time were used for making synthetics, Naehan 6, 7, 8, 9, with polycrossing method in 1997. Field experiments were carried out to compare the mophological and agronomical characteristics and forage productivity and quality of the synthetics with those of Italian ryegrass varieties, Barmultra and Hwasan 101. in Suwon and Yonchun from 1999 to 2000. Heading time of the synthetics were 22th to 24th May that belong to late-mature types to be similar to that of Barmultra and Hwasan 101 in Suwon. The synthetics were 101 to 106 c3n in plant length, medium or thick in thickness of stem, dark peen in leaf color, broad and long in flag leaf, strong in lodging resistance, and excellent in regrowth. Winter survivals of the synthetics were no different from that of Barmultra or Hwasan 101 in Suwon, but better than that of Barmultra or Hwasan 101 in Yonchun where was -10 to -12$^{\circ}C$ of minimum average air temperature in January or February. Dry matter(DM) yields of the synthetics were similar to DM 8,238kg per ha of Barmultra in Suwon, but in Yonchun, were more 7 to 13% than DM 7,291kg per ha of Barmultra. Forage qualities, IVDMD, ADF, NDF and TDN of the synthetics were lower than those of Hwasan 101, but higher than those of Barmultra.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.103-113
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• 2003

This study was conducted to investigate the optimal condition for produce of large quantity recipient oocytes on porcine cloned embryos. In order to determined the optimum concentration and exposure time of ethanol, $Ca^{2+}$-ionophore, 6-DMAP and cycloheximide, in vitro matured oocytes were activated in TCM-199 containing various chemicals and 15% FBS. The activated oocytes were cultured in Whitten's medium containing 10% FBS at 5% $CO_2$. 1. When the porcine oocytes were activated with the ethanol, the best pronucleus formation, cleavage, and in vitro development rate were obtained in the 10% for 10 minutes, which was significantly higher than all of the other treatment(53.4%, 51.6% and 39.9%, respectively). 2. When the porcine oocytes were activated with the $Ca^{2+}$-ionophore, the pronucleus furmation, cleavage, and in vitro development rate were found significantly higher in the 25$\mu$M fur 2min. treatment than those of other concentration and exposure time(59.7%, 62.2% and 43.9%, respectively). 3. When the porcine oocytes were activated with the 6-DMAP, the best pronucleus formation, cleavage, and in vitro development rate were obtained in the 2mM for 2hr~4.5hr(57.3%, 58.4% and 29.0%, respectively). 4. When the porcine oocytes were activated with the cycloheximide, result showed that pronucleus formation, cleavage, and in vifro development rate were 52.1%, 47.7% and 31.8%, respectively, in the 5$\mu\textrm{g}$/ml for 4hr~6hr treatmrent, which was significantly higher than all of the other treatment. These results suggested that the active condition of porcine oocytes was established by optimum concentration and exposure time among different chemicals for produce of large quantity recipient oocytes.s.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.127-132
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• 2004

This study was conducted to examine the effects of electric stimulation conditions on in vitro developmental ability of caprine embryos after somatic cell nuclear transfer. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean native goats. The caprine ear cells were cultured in vitro in serum-starvation condition (TCM-l99 + 0.5% FBS) for 3 to 5 days of cell confluence. The zona pellucida of in vivo and in vitro matured oocytes were partially drilled using laser system. Single somatic cell was individually transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol. After the electofusion, embryos were activated by electric stimulation or Ionomycin + 6-DMAP. Nuclear transfer embryos were cultured in mSOF medium supplemented with 0.8% BSA 6∼7 days at 39 , 5% $CO_2$, 5% $O_2$, 90% $N_2$. The fusion rate of donor cells was 60.4% and 40.3 % in ear cell and fetal fibroblast, and cleavage rate were 40.6% and 48.2%, respectively. No significant difference was found in the fusion and cleavage rate in different donor cells. Nuclear transferred oocytes were fused by electric pulses of 1.30∼1.40, 2.30∼2.39 and 2.40∼2.46 ㎸/cm. There was no significant difference among different electric pulses in fusion rates (26.7, 34.8 and 43.8%). The cleavage rate was higher (p<0.05) in 1.30∼1.40 ㎸/cm (82.9%) than 2.30∼2.39 ㎸/cm (43.8%) and 2.40∼2.46 ㎸/cm. (51.8%). The fusion rates of recipient oocyte source were 1st (43.5% and 23.6%), 2nd (55.7％ and 39.2%) and 3rd (66.1% and 52.8%) in in vivo and in vitro oocytes. However, fusion ratee were significantly higher (p<0.05) in in vivo than in vitro oocyte. The cleavage rate of fused oocytes from in vivo and in vitro sources were 52.6% and 54.4%, respectively. No significant difference was found in the cleavage rate according to the recipient oocyte source. These results suggest that factors such as field pulse of electric stimulation and oocyte source could affect in vitro developmental ability of nuclear transplanted caprine oocytes.