不同直径山羊卵泡卵母细胞的发育特征及体外发育能力

目的研究山羊卵巢表面不同直径卵泡卵母细胞的发育特征及体外发育能力,优化山羊早期胚胎体外生产系统。方法收集繁殖期和非繁殖期山羊卵巢,采集表面直径小于1.5 mm、1.5-2.5 mm、2.5-3.5 mm和大于3.5 mm4种卵泡卵母细胞,以Hoechst33342染色检查核发育阶段;同时,利用体外培养方法观察不同直径卵泡卵母细胞的成熟、受精和早期胚胎发育能力。结果直径小于1.5 mm卵泡卵母细胞主要处于GVI期;1.5-2.5 mm卵泡卵母细胞以GVⅠ、GVⅡ和GVⅢ期为主;2.5-3.5 mm卵泡卵母细胞在GVⅡ到GVⅣ期间平均分布;大于3.5mm卵泡卵母细胞主要为GVⅢ到GVBD期。体外培养实验发现,直径小于1.5 mm卵泡卵母细胞仅有个别能完成成熟和卵裂;大于1.5 mm卵泡卵母细胞具有核成熟能力,能完成成熟和受精,但1.5-2.5 mm卵泡卵母细胞的受精卵通常阻滞于4-8细胞期;当卵泡直径大于2.5 mm时,卵母细胞才能较好地支持胚胎继续发育,其桑/囊胚的比例达到30%以上。卵泡卵母细胞的发育特征和体外发育能力与动物所处的繁殖季节无关。结论山羊卵巢上直径大于1.5 mm卵泡卵母细胞具有核成熟能力,大于2.5 mm卵泡卵母细胞能支持早期胚胎继续发育。

Developmental Characteristics of Goat Oocytes from Different Diameter Follicles and Their Competence of Maturation,Fertilization and Culture in Vitro

Objective To improve the efficiency of embryo production in vitro,developmental characteristics of goat oocytes from different diameter follicles and their competence of maturation,fertilization and culture in vitro were investigated.Methods Oocytes were collected by dissection of surface-visible follicles from slaughterhouse goat ovaries,and they were divided into four groups base on the follicle diameter： 〈1.5 mm,1.5～2.5 mm,2.5～3.5 mm and 〉3.5 mm.Developmental characteristics of the oocytes nuclei were assessed using Hoechest33342 staining before culture.The developmental competence was compared after in vitro maturation（IVM）,in vitro fertilization（IVF） and in vitro culture（IVC）.Results More than 85% oocytes from 〈1.5 mm follicles were at germinal vesicle phase I（GVⅠ）.A majority of oocytes from 1.5～2.5 mm follicles had developed to GV Ⅱ or GV Ⅲ（above 70%）.Almost all oocytes from 2.5～3.5 mm follicles developed beyond GV Iphase and into GV Ⅱ～GVBD.When the diameter of follicles was larger than 3.5 mm,aproximately 75% of the oocytes developed to GV Ⅲ,GV IV or GVBD.After maturation,only about 10% of oocytes from 〈1.5 mm follicles developed to metaphase Ⅱ（M Ⅱ）,significantily lower than that in other three groups（55%～80%,P〈0.05）.After fertilization and culture,no significant difference in cleavage rate was observed among the three groups in which oocytes originating from follicles 〉1.5 mm.However,the developmental competence of fertilized oocytes rose as the follicle diameter increased.In 1.5～2.5 mm group,about 10% of fertilized oocytes reached morula and blastocyst stage,siginificantly lower than that in 2.5～3.5 mm and 〉3.5 mm groups（31.3%～33.3% and 41.7%～47.4%,respectively）.Developmental characteristics of oocytes and their competence of IVM,IVF and IVC were independent of breeding or nonbreeding season.Conclusion Oocytes only from follicles 〉1.5 mm are able to mature to M Ⅱ,and full developmental competence has acquired in oocytes