| Primary influence factors within OPU technique were experimented, and the methods of improving the development of early embryo were also researched in this study for the sake of establishing an acceptable OPU-IVF system on holstein cow. The main contents of this study are: effcet of primary factors on cow OPU technique, the methods of improving the development of early embryo and in vitro production of embryo on cow.In the study of cow OPU technique, the 4 different aspiration pressure (70, 90, 110, 130mmHg) , the different schedule and prior to adiministration of FSH were experimented. The results shows, the recovery rate and quality of COCs can be influenced by aspiration pressure: the numbers of oocytes per donor cow obtioned with the 70mmHg pressure (4.35 + 2.03) were lower than the else groups (P<0.05) and there is no siginificant difference between the 90mmHg, HOmmHg and 130mmHg. The recovery rate under 70mmHg pressure (44.16%) is significant lower than 90mmHg pressure (56.04%)(P<0.05), the recovery rate is no significant difference between HOmmHg and 130mmHg, but significant higher than 70mmHg and 90mmHg (66.03%, 67.29% respectively)(P<0.05). Under the 130mmHg pressure, the recovery rate of grade A and B (48.61%) is significant lower than the other groups (P<0.05), and there are no significant difference between the other groups. Consider the recovery rate and quality of oocytes, HOmmHg is the best aspiration pressure under our lab condition (the recovery rate is 66.03%, and the rate of grade A and B is 68.84%). The interval of OPU influence the number of follicles available for puncture, the numbers of oocytes per aspiration and the recovery rate: with the 3 day's interval, 7.71 + 1.51 follicles were punctured and 5.29 + 1.19 oocytes were recovered in per aspiration, this is significant lower than first OPU, 7 day's interval and 14 day's interval;but the recovery rate (68.61 +4.19%)is higher than the other groups (P<0.05). Of all groups, more oocytes can be retrived during the same period (10.58 oocytes/w) with the schedual of 3 day's interval. FSH stimulation prior to OPU increased the number of follicles available for puncture to 12.36 + 2.39, increased the oocytes retrieval rate to 8.60+1.85 oocytes per aspiration, this is significant higher than non stimulated group(P<0.05). There is no significant difference between the stimulated and non stimulated groups in the rate of COCs, cleavage rate and morula-blastocyst development rate(75.19% , 58.77% , 27.61%).In the study of improving development capacity of early embryos, the effects of different IVC media were experimented. The IVMD-101 (Japan) was superseded byTCM-199+BSA+Pyruvate+ITS+ab, the morula rate (25.16%) and the blastocyst rate (17.14%) were significant higher than IVMD-101 media (12.26% and 8.49% , respectively);The optimum concentration of ITS is 1 Oul/ml.In the study of IVP of cow, the oocytes retrieved from OPU were matured in vitro to the morula and blastocyst stage. There are two kinds of sperms were used in fertilization. The result shows that the sex controled sperm can be used to IVF successfully, and the morula rate is 27%, the blastocyst rate is 18%, this is similar to the routine sperm.In conclusion, it's feasible to apply OPU and IVF technique to IVP. In this study, 12 OPU were implemented, 7.5 oocytes were retrieved and 1.2 blastocyst were gained in per aspiration. |
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