Study On Different Techniques Of Dairy Embryo Transplantation And Establishment Of Production System

Embryo transfer in cattle mainly includes Estrus synchronization of recipients and donors, superovulation embryo production ,cryopreservation and embryo transfer. The key techniques of bovine embryo transfer were systematically studied , and some relevant techniques such as OPU (Ovum Pick-Up), in vitro fertilization (IVF) and embryos sexing ,etc. were performed in this thesis. The main research contents in this paper are : 1.Study on estrus synchronization in cattle; 2. Study on superovulation in cattle; 3. Study on main factors affected on embryo transfer in cattle; 4.Applied study on OPU and IVF in cattle ; 5. Applied study on embryo sexing in dairy cattle. The results were as following:Estrus synchronization: Several methods such as vaginal device containing progesterone (P₄)plus injection of prostaglandin (PG) were used to induce synchronous estrus in different condition of cattle. The synchronized estrus results were analyzed and investigated according to estrus rate, available CL rate, available recipients rate, pregnancy rate and obtained transferable embryos number, etc. The research results showed that induced estrus with use of different kinds of methods could substitute natural estrus. The method induced estrus with use of vaginal device containing P₄ was not restricted by estrus cycle ,which the treatment could be done at any time according to production and scientific research plan ,the effects of the method with use of sponge plug made in China and imported CIDR on induced estrus results were not significantly different. The cost of sponge plug made in China was decreased by 70%, therefore, the sponge plug plus injection of PG is better to be used for inducing estrus synchronization in cattle; the effect of induced estrus in higher nutrition level of cows herd was significantly better than other kinds of cows herd. This conclusion is very important for selection of donor cows and recipient cows in embryo transfer. The rate of synchronous estrus of heifers was significantly higher than that of multiparous cows , however, the rate of available CL of both were not significantly different ; The rate of multiparous cows nursing calf more than 3 months were significantly higher than that of less than 3 months.Superovulation : The response of beginning superovulation treatment with Folltropin-V was the best on the day 9 folowing spontsneously estrus ; which mean fertilized ova and transferable embryos were 12 and 6.8 , respectively, significantly higher than those from the cows treated on the day 3, day 6 and day 12 (p<0.05); there were no significant difference in the superovulatary responses among the groups of 4-day constant injection and decreasing injection ,and 5-day constant injection and decreasing injection(p>0.05); the superovlutary result obtained with the vaginal device (such as CIDR and Sponge plug) combination with injection of E₂+P₄was significantly higher than that treated with the vaginal device alone or the device +P₄ only (p<0.05),which indicated that the estradiol (E₂) could promote superovulation obviously; there were no significant difference in the supeovulatary results between the groups treated with the vaginal device +E₂+P₄ and traditional method (beginning FSH treatment on the day 8 to day 9 following spontaneously estrus) not only in the beef cows but also in dairy cows (p>0.05); There were no significant difference in the superovulatary responses between the groups treated with imported CIDR+E₂+P₄ and "Niuhuan "Sponse+E₂+P₄ in heifers or cows (p>0.05); the vaginal sponge plug "Niuhuan" ,made in China, could replace the imported CIDR, and the cost could decreased about 70% compare to CIDR, the times of short term repeat superovulation treatments (25days -28 days interval) could not influenced superovulatary results significantly (p>0.05)in Hostein dairy cows ,but, compare to the traditional method ; the superovulation method following induction of synchronous follicular wave occur with use of vaginal divice plus injection of E₂ and P₄ could increase the repeatedtreatment times, which could enhance superovulatary efficiency and total embryos production annually. The superovulation treatment times could be up to 10 times each year, and the number of transferable embryos recovered per donor could be over 60 annually. However, traditional method could repeat only about 4 times, and total transferable embryos recovered per donor was about 24 each year. Embryo transfer: The factors of affecting on pregnancy rate of embryo transfer were investigated in this study. The pregnancy rates of recipients were not affected by the embryos frozen inl.5M Ethylene Glycol (EG),10% Glycerol (G) or EFS40 , the embryos frozen in Ethylene Glycol (EG) could be directly transferred after thawing, which could be spread in large scale. The pregnancy rate of morulae was slightly higher than that of early blastocystes or blastocystes ,but there were no statistical differences among the three kind of embryo stages (>0.05) ; 12 hours earlier or later asynchrony between the age of the embryo and the estrus cycle of the recipients; embryos could not affect pregnant rate of embryo transfer; but the rate could decreased remarkably if morulae transferred into the 6 d recipients. There were no significant difference in the rate of pregnancy rate among the semi-embryos, double semi-embryos intact embryos transfer (P>0.05), however, there were significant difference among the three groups (P>0.05), the semi-embryos could increase the embryos utilization rate; The pregnant rate of heifers was higher than that of cows, but not significantly different; The pregnant rate of recipients raised under typical pasture condition was significantly lower than that of feeding more supplement under pasture condition and standard fed in feedlot (P<0.01) ,there were no significant difference between the later groups (P>0.05) Different breed of recipients could also affect the pregnancy rate of embryo transfer, the pregnant rate of Yellow Cattle recipients was lower than that of dairy cattle as the embryo transfer recipients (P<0.05).OPU and IVF: No matter the heifers or cows, the number of oocytes collected twice a week was higher than that of oocytes collected once a week (P<0.01). The number of oocytes collected twice a week was higher from cows than from heifers (P < 0.05) ; The aspiration negative pressure could influence the oocytes collection rate and proportion of good quality oocytes(P<0.01); The aspiration negative pressure of 40 mmHg was better for OPU. The oocytes could be collected from aged cows, early pregnancy cows and the cows with reproductive diseases, etc. with use of OPU, but the number of oocytes collected from the cows was significantly lower than that collected from the healthy cows; Hormones treatment could enhance follicle development before OPU, that could increase number of oocytes collection. In the IVF study, the maturation rate and fertilization rate of oocytes cultured in group IVMD-101 were remarkably higher group TCM-199 (P<0.01) . group could promote not only oocytes maturation rate but also fertilization rate.After maturation in IVMD-101,the maturated oocytes were fertilized in BO medium and IVF-100 medium, the results of IVMD-101 and SOFaa for development culture were similar, the rates of morulae and blastocystes were not significantly different between the two groups of development media, but remarkably higher than the group of TCM-199 for development medium (P<0.05 )Embryo sexing: The pregnancy rate of the fresh biopsied embryos implantation by cutting method was 50%, and the rate of aspiration method was 57.6%, there were no significantly differences between them(P.0.05). Cutting embryos as a embryo biopsy is a convenient method, aspiration ,however, need relatively higher skills and very difficult to be spread in production, although some researchers considered that it was less damage to the embryos. Lamp method could be used as embryo sexing, it was fast and correct method for embryo sexing , the correct rate of embryo sexing by Lamp method was up to 97.9%, its cost was lower than PCR method.