| It is the development that the bovine oocyte can be researched to produce embryos in vitro for studying and demonstration. Bovine embryo production includes oocyte in vitro maturation, in vitro fertilization, in vitro culture etc. The article had reported how to carry out the process of oocyte in vitro and had got more dates and materials for the future studies.1 Bovine Oocyte in Vitro MaturationThe Effect of in Vitro Maturation with Oocyte GradeThere were four COCs grade in the study. The maturated oocyte rate were 63.81% for grade A,47.81% for grade B, 2.01% for grade C and 0.00% for grade D. The difference among four grades were very significantly(p<0.01)each other. Grade A and B of COCs were significantly higher than grade C and D. The grade A and B of COCs would be chose to maturate in following system.The Effect of in Vitro Maturation with Different Diameter FolliclesThere were three kind of follicles: big follicle(sφ>8mm),middle follicle(s2mm≤φ≤8mm) and small follicles(φ<2mm). The maturated oocyte rate for different diameter follicles were 17.07% for big's, 61.82% for middle's and 19.06% for small's. The middle's was significantly higher than big's and small'(sp<0.01).There were not significant difference between big's and small's(p>0.05). It had showed that the follicular diameter was very important for in vitro maturation of COCs. The best choice was to collect middle diameter follicles to do.2 Bovine Oocyte in Vitro FertilizationThe Effect of Sperm Capacitation and Oocyte Fertilization Rate by Direct Centrifuge to Select Sperm.Result for second centrifugal sperm for the deposit-liquid which was first centrifuged.After the second sperm centrifuged and capacitated, the sperm washed activation was 0.44, the high sperm capacitation was 0.41 at 10min and 0.32 at 15min, the super activation showed at 15min, and the fertilization rate of oocyte was 40.19%.Result for second centrifugal sperm for the up-liquid which was first centrifuged.After the second sperm centrifuged and capacitated, the sperm washed activation was 0.56,the high sperm capacitation was 0.59 at 10min and 0.585 at 15min, the super activation showed at 15min, and the fertilization rate of oocyte was 43.21%.It were made know that the sperm activation, super activation and oocyte fertilization rate of up-liquid of second centrifuged were significantly higher than the deposit-liquid of second centrifuge's(p<0.01).The up-liquid's were also observed that had clear field of microscope, had good diaphaneity, had not eyeable cryoprotectant and dilutent, had few died and abnormal sperms. The deposit-liquid's were observed that had not clear field of microscope, had not clear diaphaneity, had much eyeable cryoprotectant and dilutent, had many died and abnormal sperms. It is a new method to wash and capacitate sperm with two step by centrifugating for the first up-liquid.The Effect of Sperm Capacitation and Oocyte Fertilization Rate under the Different Temperature by Swimming-upSwimming-up in room temperature(20～24℃).The thawing frozen sperm had been swum up 60min on the super clear desk in room temperature. It was indicated that the best sperm activation was 0.68 at 45min, the best sperm super capacitation was at 15min after treatment, and the fertilization rate was 41.40%.Swimming-up in constant temperature(38.5℃).The thawing frozen sperm had been swum up 60min in CO2 incubator in constant temperature at 38.5℃. It was indicated that the best sperm activation was 0.697 at 30 min, the best sperm super capacitation was at 15min after treatment, and the fertilization rate was 41.51%.The sperm activation and fertilization rate were not significantly difference between the two treatment. But the constant temperature group was better than the room temperature group. It was knew that the room temperature was not very stability and always changing along with the environment temperature. And the sperm activation and capacitation was also instability. Swimming-up in constant temperature would be the best way to select sperm because it was easy to control and stability for the sperm activation and capacitation.The Sperm Concentration for Oocyte Fertilization RateThe study was conducted to optimize the fertilization condition. Sperm concentration had great effect on the oocyte fertilization rate. It were showed that the oocyte fertilization rate of middle sperm concentration group(1.5×106/ml, 47.34%)was significantly higher than the high sperm concentration group(3.0×106/ml, 33.96%)and low sperm concentration group(1.0×106/ml, 42.38%)(p<0.01). And the low group's was significantly higher than the high group's(p<0.01). The sperm concentration would be applied at (1.0～1.5)×106/ml and had less abnormal sperm and increased the fertilization rate.3 Bovine Oocyte in Vitro CultureIn Vitro Culture with Co-culture SystemThere were 3 co-culture system groups in the experiment.All of them were cumulus, cumulus+bFF and control group. The morula rate of its were 29.07%,34.99% and 26.67% and the blastocyst rate of its were 8.51%,10.63% and 7.24%.The morula and blastocyst rate of cumulus+bFF group were significantly higher than the cumulus and control group(p<0.01).There was 2-cell block in the field for oocyte embryo development.Co-culture with cumulus+bFF played a important role for embryo development to blastocyst and had overcome the 2-cell block.Oocyte Embryos Development with Medium VolumeIt were treated that maturated oocyte were cultured with 50μl and/or 500μl medium volume in 4-hole culture plate. The morula and blastocyst rate of 50μl medium were 33.14% and 9.07%. The morula and blastocyst rate of 500μl medium volume were 36.23% and 10.23%. The two rates of 500μl medium volume were significantly higher than the 50μl medium's(p<0.01).It was reported that the small medium volume would not maintain the minienvironment verywell because it was easy to change, but the large medium volume as 500μl would maintain the minienvironment well and had had embryo development.4 Freezing of COCsOocyte been Frozen with 10%GL and 10%EG of Cryoprotectant by One-way ProcessHigh grade COCs was frozen and stored following the designed process and thawed one week late. The maturation and morula and blastocyst rate of 10%EG group were 34.46%,11.18% and 4.18%, and 10%GL group's were 33.44%,10.56% and 3.86%.The difference of all between the two groups was not significantly(p>0.05).The trial had evidenced 10%GL and 10%EG of cryoprotectant adapted to freeze and store COCs and had the same function.Oocyte been Frozen for Carrier by VitrificationHigh grade COCs was also frozen and stored following the designed process and thawed one week late. The maturation and morula and blastocyst rate of wheat straw group were 29.84%, 9.34% and 3.47%, and OPS group's were 31.89%, 9.66% and 3.80%. The difference of all between the two groups was not significantly(p>0.05). The results had showed that wheat straw and OPS of carrier adapted to freeze and store COCs, and had the same function.Comparing with the Frozen WaysAccording to above exprements of COCs frozen, the effect of one-way process was better than the vitrification process. The predominance sequence of frozen effect could express as follow as 10%EG group>10% GL group>OPS group>wheat straw group.5 Brief Summary.It was carried out that the bovine oocyte in vitro production and freezing were studied. Through this series experiments it had been learned and applied how to maturate oocyte in vitro, to capacitate sperm, to fertilize oocyte in vitro, to freeze oocyte by one-way or vitrification process, and to culture thawing oocyte been frozen. The 2-cell block had been overcomed by co-culture system with cumulus or cumulus+bFF. One new centrifugal method for sperm capacitation had been tested and applied in this research and got better than before.
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