The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199 + FCS with various concentrations (0-20 mu-M) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 mu-M nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 mu-M nocodazole. Exposure to 10 mu-M nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 mu-M nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% O2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 mu-M nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 mu-M nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used-for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. IN experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres no treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres oat 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P<0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P<0.05) and development to the blastocyst stage (P<0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion,degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P0.05) was obtained by increasing pulse strength from 30 to 300 V/mm. The embryos fused at the pules strengths of 30 to 70 V/mm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P0.05). There were no significant differences in the rates of fusion, degeneration and d development to blastocysts when the pulse duration was increased from 30 to 90 mu-sec. Although fusion rates were increased (P0.05) by increasing the pulse number up to 4, a significant decrease (P0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 7- V/mm, 70 mu-sec pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P0.05)

This study ws carried out to determine the effects of supplementation of the maturation media with insulin on in vitro maturation and fertilization of bovine oocytes. In Experiment 1, cumulus-intact bovine oocytes were cultured in a maturation medium (TCM-199 containing 10% fetal calf serum, 0.02 U/ml follicular stimulating hormone and 1 mu-g/ml estradiol-17beta) with or without insulin supplementation (10 mu-g/ml). The maturation and fertilization rates of oocytes and subsequent embryonic development to the blastocyst stage were not affected by the treatment with insulin in the presence of serum and the hormones during the maturation period. In Experiment 2, to avoid the effects of serum and the hormones, a serum- and hormone-free maturation medium (TCM-199 containing 1 mg/ml polyvinyl alcohol) was used. In the absence of serum and hormones during the maturation period, the maturation rate was not affected by treatment with insulin, but the fertilization rate was improved. In Experiment 3, when denuded oocytes were inseminated together with cumulus cells cultured in serum- and hormone-free maturation medium supplemented with insulin, the fertilization rate was increased. These results demonstrate that the addition of insulin to the serum- and hormone-free maturation medium improves the fertilization rate of bovine oocytes in vitro, and suggest that insulin may stimulate the secretion of sperm capacitating agent(s) from cumulus cells