【作者】 吴国利；

【导师】 赵德明；

【作者基本信息】 中国农业大学 ， 临床兽医， 2005， 博士

【摘要】 牛胚胎移植主要包括:供受体同期发情、供体超数排卵、胚胎冷冻解冻和移植等关键环节,本文通过对影响这些关键环节的技术进行对比研究,进而探讨适宜的生物技术,为奶牛胚胎移植产业化提出高效能、低成本、稳定和操作简便的技术体系;对一些相关的胚胎生物技术,如活体采卵、体外受精、胚胎性别鉴定等技术进行了研究探讨。 本文研究的主要内容:(一) 诱导奶牛同期发情试剂和相关技术的对比研究。(二) 提高牛超数排卵效果技术研究。(三) 牛胚胎移植的主要影响因素研究。(四) 活体采卵(OPU)及体外受精(IVF)技术的应用研究。(五) 奶牛胚胎性别鉴定技术的应用研究。 诱导奶牛同期发情试剂和相关技术的对比研究:用含孕酮阴道栓、PG等几种方法,对不同状况的牛群进行同期发情处理,根据发情率、黄体合格率、受体移植可用率、妊娠率以及超数排卵获取的可用胚数,进行发情程度、效果的对比分析研究。研究结果表明:不同方法的诱导发情完全可以替代自然发情,孕酮阴道栓加PG诱导发情的方法不受发情周期的限制,可以随时进行处理,国产的孕酮海绵栓和进口的CIDR对发情程度的影响差异不显著,用国产孕酮海绵栓完全可以替代进口CIDR,成本下降70%左右:在不同的牛群,营养状况水平较高的牛群同期发情水平极显著高于其他牛群,本结论对胚胎移植的供、受体牛的选择具有重要意义。产犊超过3个月的经产牛的同期发情率都显著高于产犊不足3个月的经产牛的同期发情率(P<0.01)。 提高牛超数排卵效果技术研究:在自然发情周期的第9天,开始注射FOLLITROPIN-V超排处理,超排反应最佳,按不同时间注射相同剂量FOLLITROPIN-V,在第9天注射所获得的受精卵每头平均12枚、可用胚每头平均6.8枚,显著的高于3天、6天、12天所获得的数量(P<0.05); 应用含孕激素阴道拴+雌激素+孕激素处理后,超排效果显著高于单独使用阴道拴组和阴道拴配合孕激素组,说明雌激素对超排具有明显促进作用;应用阴道拴配合雌激素和孕激素处理后超排,与在发情周期的第8-12天开始的传统超排方法比较,结果差异显著(P<0.05);应用进口阴道拴CIDR和国产“牛欢”(海绵阴道栓)配合雌激素和孕激素处理后开始超排,对青年牛和成母牛超排反应均没有差异(P>0.05),国产的阴道海绵栓可以代替进口的CIDR,成本下降70%左右;短期重复超排(间隔25天-28天)处理次数对超排效果与传统方法比较差异不显著(P>0.05),应用阴道拴诱导卵泡波同步发生后开始超排处理后可以提高重复超排处理次数,提高超排效率,增加胚胎年产量,采用重复超排技术,每年超排次数可高达10次,每头牛可年产达60枚以上,而传统方法只能达到4次左右,所获得的可用胚胎也只有24枚左右。 本文就影响牛体内胚胎移植妊娠率的主要因素进行研究分析,结果如下:1.5M乙二醇,10%的甘油和EFS40(玻璃化冷冻液)三种不同的胚胎冷冻法对受体的妊娠率没有明显的差别。在牛胚胎移植产业化中可相互补充,尤其乙二醇作为冷冻保护液,可以达到解冻后直接移植的目的,简化操作,便于大规模推广应用;致密桑椹胚的移植妊娠率略高于早期囊胚和囊胚,但三者的移植妊娠率差异不显著(P>0.05);胚胎与受体的同步差±12小时内不影响受体移植妊娠率,但更多还原

【Abstract】 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.更多还原