| 1. Effects of cycloheximide (CHX) and cytochalasin B (CB) on the parthenogenetic development of buffalo oocytes were investigated. Buffalo oocytes matured in vitro for 27~28h were treated with 7% EH for 5 min, then cultured in the media containing different concentration of CHX (0, 0.625, 1.25, 2.5, 5ug/ml) for 10h, and then fixed for staining or cultured in CM for checking cleavage. The percentage of oocytes cleaved and formed pronucleus in either 0.625 (Ag/ml or 1.25ug/ml CHX group was significantly higher than the control group (P<0.05). However, when the concentration of CHX was increased to 5 ug/ml, the cleavage rate and pronuclear formation rate were decreased significantly (P<0.01). Addition of 3ug/ml CB to CM containing 0.625 ug/ml CHX increased the proportion of oocytes with two pronuclei (38.9% vs 15.1%, P<0.05) and developed into morulae/blastocysts (40.7% vs 26.8%, P<0.05), but did not affect the percentage of oocytes cleaved and total pronucleus rate. These results indicate that CHX at an appropriate concentration can improve the parthenogenetic development of buffalo oocytes, CB at an appropriate concentration may inhibit the extrusion of the first polar body and increase the proportion of activated oocytes with two pronuclei.2. Methods for activating buffalo oocytes were studied. Buffalo oocytes matured in vitro for 26h were activated by 7% ethanol (EH), A23187 or lonomycin (Ion) for 5min, then cultured in culture medium (CM) containing 2mmol/L for 3-4h, and then cultured in CM for 6-lldays to evaluate embryos development. The cleavage and pronuclear formation rate of oocytes treated with 5umol/L Ion were significantly higher than that of oocytes with EH treatment. Exposing buffalo oocytes to free Ca2+ medium prior to activation treatment did not affect their parthenogenetic development. When buffalo oocytes are activated by A23187, the cleavage increased as the concentrations of A23187 increased. These results indicate that ionomycin can activate buffalo oocytes effectively.3. Effects of in vitro maturation (IVM) duration and enucleation time of buffalo oocytes on the NT embryo development were studied. The maturation rate ofoocytes matured for IVM16-18h was lower than that of IVM for 19-21h (43.0% vs 58.2%, P<0.05), and was significantly lower than that of IVM for 22-24h (43.0% vs 68.8%, P<0.01). However, the enucleation rate decreased as the IVM duration prolonged. There were no significant difference in fusion rate, cleavage rate and blastocyte development rate with different enucleation time of oocytes during IVM16-24h (P>0.05). The results indicate that maturation rate of buffalo oocytes increase as the IVM duration prolongs, while the enucleation rate decreases, however, which has no significant difference on development of NT embryos with different enucleation time of oocytes during IVM16~24h.4. The possibility of adjusting enucleation time by 6-DMAP was explored. Compared with the cytoplasts matured in the maturation medium directly for 16~18h, the fusion rate, cleavage rate and blastocyst development of reconstructed embryos from oocytes cultured in 2mmol/L 6-DMAP for 3-5h prior to IVM did not differ significantly from that of reconstructed embryos from oocytes matured in the maturation medium directly for 16~18h. This result indicates that 6-DMAP may be employed for arranging enucleation of oocytes at a convenient time.5. The position of buffalo oocyte nucleolus related to its PB1 during the final stage of IVM, and the feasibility of increasing enucleation rate using Spindle-View system were studied. The space between PBi and spindle (SP) of IVM buffalo oocytes increased as the IVM duration prolonged. The SP are relatively near to PB1 when oocytes aged at 18h, 20h, 22h (72.6%, 67.9%, 64.0% respectively), when IVM time reached at 24h, the percentage decreased to 57.6%. Enucleation by Spindle-View system could eliminate the effects of changing of the position on enucleation rate. These results indicate that Spindle-View system can increase...
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