| This dissertation studed systematically how to utilize embryo engineering technology to improve reproductive efficiency of dairy cows, including the techniques of MOET, IVP, sex control and the prevention and cure of reproductive disorder. The result showed: 1. 1127 donor cows were superovulated. The total number of recovered embryos was 10900 ,available embryos 6942,and average number of available embryos 6.73 per cow per time for successful superovulation. Three protocols including PSO1, PSO2 and PSO3 were applied in this experiment and the success rates of superovulation were 89.50%, 90.02% and 98.54%, respectively. The best result of superovulation was obtained in cows when initial injection of FSH directly without pre-treatment on day 9 to 11 of spontaneous estrus. 2. The age of the first time for superovulation of heifers should be restricted and optimal age of superovulation should be above the 15 months old. The continuous repeat superovulation to donor cows should be less than 3 times because the proportion of available embryos decreased evidently after treatment over 4 times. In addition, the interval between two superovulations should be 46 to 60 days. In multiparous cows, 1 to 3 parities were optimal to superovulation and it was not suitable for cows more than 7 parities. The interval between parturition and superovulation should be 80 to 90 days. 3. Superovulated donor cows were inseminated artificially by sexed semen. The average number of recovered embryos and available embryos was 9.02 and 4.70, respectively. The ET pregnancy rate of sexed embryos was closed to that of normal embryos. The result suggested that the sexed embryo might be produced by sexed semen. 4. In the study, 5083 recipient cows were treated by three synchronization procotols: double PG injection, CIDR+PG treatment and the single injection of PG. The synchronization rates were 75%, 78.33% and 72% correspondingly. The pregnancy rate of fresh embryo was 55% after embryo transfer, and that of frozen embryos was 46~47%, the result of the three groups was closed. The utilization rate of heifer recipients was higher than multiparous recipients after treatment of estrus synchronization. It was concluded that the protocol of double PG injection was better than the two others. However, the recipient utilization rate of spontaneous estrus was 85%, significantly higher than the double PG injection group, so the success rate of embryo transfer can be remarkably improved by utilizing recipient of spontaneous estrtus. 5. The frozen embryos were thawed by direct and programed methods, the pregnancy rate after embryo transfer was 45.22% and 47.64%, respectively. The result showed that the embryos frozen by EG as freezing medium might be directly transferred after thawing in 35℃. 6. The recipient utilization rate and the pregnancy rate of nonpregnancy cows were improved after adjusting the reproductive functions with Chinese traditional medicine before synchronization. The utilization rate of parturient recipient cows treated by GnRH combined with PG during 20 to 25 days postpartum was closed to that of the natural recovered parturient cows, the interval between parturition and synchronization could be shortened by treatment of GnRH with PG. 7. The in vitro maturation rate, fertilization rate and blastocyst rate of oocytes from follicles with diameter of 3 to 6 mm were higher than that from others. Adding EGF 10 ng/mL to maturation medium benefitted the nuclei and cytoplasm maturation. In the process of the oocyte maturation, at least three-layer granule cells were necessary, and it could improve the sperm capacitation and fertilization. 8. In the sperm capacitation processes, the treatment time of Percoll method is shorter and the number of survival sperm is higher than that of swim-up method, but frequent polysperm was occurred. While swim-up method recovered few sperm but sperm viability is higher than Percoll method. Adding caffeine to capacitation medium had no significant effect on viability and fertilization capacity of sperm, even decrease the survival time of sperm. 9. The blastocyst rate was 12.0% and expanded blastocyst rate was 0% in protein-free medium (added PVA), the blastocyst rate and expanded blastocyst rate in media added BSA and FBS were 25.6%/28.0% and 10.0%/19.8%, respectively. Adding 2%Neaa and 1%Eaa to medium can significantly improve the development potential and the embryo quality. The embryos cultured in CR1aa+ 0.3%BSA medium for three days, then in SOFaa+5%FBS medium can get higher blastocyst rate and more embryo cells. Adding IGF-I to medium can improve the expanding potential of blastocyst. The co-culture system could improve the development potential of embryos (2.0% vs. 39.0%). 10. The fertilization rate of sexed sperm is slightly lower than that of the normal sperm when used for IVF, but there was no significant difference to the development potential of embryos between two groups. 127 sexed IVF embryos were transferred to 107 recipient cows, the pregnancy rate was 36.5%. 11. 998 in vivo embryos (including 136 fresh embryos and 835 thawed embryos) from normal sperm ,10 in vivo embryos from sexed sperm, 235 in vitro embryos from normal sperm and 116 in vitro embryos from sexed sperm were identified by consecutive and multiplex PCR. The pregnancy rate of identified in vivo fresh and thawed embryos from normal sperm was 41.03% and 37.72%,respectively, and from sexed sperm was 40%. The pregnancy rate of identified in vitro embryos from normal and sexed sperm was 27.78% and 25.00%, respectively. The coincident rate between the birth gender and identificationresult was 97.85%. 12. Ovarian cysts are defined as anovulatory fluid-filled cystic structures ≧?25 mm in diameter that persist on the ovaries for more than 10 days. The cure rates of hormonal therapy with single GnRH injection, GnRH+PG injection, one treatment period of orally traditional Chinese medicine, GnRH+traditional Chinese medicine were 83.0%, 86.4%, 73.8% and 87.4%, respectively. The pregnancy rates by AI of cured cows of first estrus were 52.3%, 54.4%, 58.1% and 55.3%, respectively. 13. After two courses of treatment with the traditional Chinese medicine, the total cure rate of donor cows with hard tumidness formed from incomplete corpus luteum degeneration after superovulation was 91.3%. When cured cows were superovulated again, there was no significant difference of average available embryo number and available rate between cured cows and normal cows. Pregnant-urge perfusion fluid and hormonal therapy with PG injection for persistant corpus luteum had the best effect. The cure rates were 85.9% and 89.1% and the pregnancy rate by AI after two estruses of cured cows was 77.6% and 75.5%, respectively. 14. Four treatment groups of hypovaria were FSH+LH group, PMSG+hCG group, pregnant-urge perfusion fluid group and the traditional Chinese medicine taken group. The recovery rates were 75.6%, 63.8%, 73.8% and 82.5%, respectively, and the pregnancy rates of cured cows after two estruses were 62.9%, 58.2%, 65.7% and 67.3%, respectively. 15. The terramycin oil and pregnant-urge perfusion fluid were preferably for treatment of chronic endometritis in this experiment. The cure rates were 85.4% and 84.8%, and the pregnancy rates by AI of cured cows after two estruses were 83.9% and 82.0%, respectively. The cure rates of PG injection group and traditional Chinese medicine taken group were 76.3% and 79.4%, and the pregnancy rate by AI of cured cows after two estruses was 78.9% and 84.7%, respectively. 16. Three treatments were taken for prevention of embryonic loss. The pregnancy rate of the control group, P4 supplementation group, hCG group and the traditional Chinese medicine taken group after AI was 53.3%, 56.1%, 62.1% and 58.3%, respectively; the rate after embryo transfer was 43.3%, 49.2%, 53.3% and 55.3%, respectively. The pregnancy rates of the treated groups were improved. |
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