山羊体外受精胚胎序贯培养的研究

Study on Sequencing-culture of in vitro Fertilized Embryo in Goat

目的:根据早期胚胎不同发育阶段的营养需求设计体外培养体系,以建立适于山羊早期胚胎体外发育的序贯培养方法。方法:对屠宰场来源山羊卵巢卵母细胞进行体外成熟和体外受精后移入添加不同胚胎发育影响因子的培养体系中进行体外培养,显微镜观察、统计各阶段胚胎发育情况,并对培养的胚胎进行移植。结果:BSA和EGF对山羊体外受精卵具有明显促卵裂作用,培养系统中添加EGS、EGF、HTAU或β-Me可有效支持8-细胞期山羊胚胎克服发育阻滞而提高桑葚胚发育率;在不同胚胎期添加各发育影响因子进行序贯培养的卵裂率、≥8-细胞率和桑葚胚发育率分别为45.2%、60.6%和23.4%,序贯培养的胚胎移植后产羔率为11.1%。结论:宜根据早期胚胎各时期代谢特点和营养需求对山羊胚胎进行序贯培养。

Objective：The experiments were carried out to design the system of in vitro culture （IVC） according to the nutritional requirement of different - stage early in vitro fertilized （IVF） embryos, i.e. to establish the methods of sequencing - culture adapted for IVF embryos in goat. Method： Oocytes collected from slaughtered goat ovaries by aspiration method were matured in droplets and fertilized after co - incubating with spermatozoa for 16- 20 h, then tire IVF eggs were transferred into several basic cultured systems which composed by modified synthesis oviduct fluid （mSOF） and cumulus cell roonolayer （CC） and different developed factors of IVF embryos such as BSA, estrous goat serum （EGS）, epidermal growth factor （EGF）, hypotaurine （HTAU） or 19- Mercaptoethanol（19- Me）. The rate of embryonic development was evaluated throughout the culture period. Finally, the excellent embryos were transferred into recipients. Result： BSA and EGF can beneficially improve the fertilized goat embryos to cleavage, however, optimal dose of EGS, EGF, HTAU and 19 - Me in developed culture system can positively support 8 - cell stage embryos to overcome the developmental retardation and increase tire frequency of morulae formation. The cleavage rate, 1〉8 - cell rate and morula rate of the inseminated eggs derived from sequencing- culture method were 45.2%, 60.6% and 23.4%, separately. Two lambs were born after eighteen sequencing- cultured embryos were transferred into six recipients （11.1%）. Conclusion： The IVF early embryos should be sequencing - cultured according the metabolism characteristic and nutritional requirement of different developed stags embryos.