Studies on in vitro maturation, fertilization and developmental capacity of bovine oocytes cultured in intact follicles

第一年(1987)对牛卵泡内和卵泡外成熟卵母细胞的受精和卵裂进行了研究。体外受精或在兔输卵管内受精，体外受精和异种受精的卵母细胞分别在体外和体内(兔输卵管)培养后观察卵裂情况。卵泡内法与卵泡外法相比，核成熟率分别为57.9%和52.7%，差异不显著(P>0.05)。然而，兔输卵管内卵母细胞的体内受精卵(49.3%)和卵裂卵(34.6%)所占比例显著高于卵泡外卵母细胞(p&lt；0.025)。这些结果表明，牛卵母细胞卵泡内培养提供了细胞质成熟的生理环境，比传统的卵泡外卵母细胞培养具有更高的受精率和卵裂率。第二年(1988年)，建立了卵母细胞成熟的共培养体系。用上游法和肝素处理牛冷冻精子可提高体外受精率(50-60%)。目前，我已经获得了体外成熟率(60%-70%)、受精率(50%)、卵裂率(50%)和囊胚率(10%-20%)。牛输卵管上皮细胞成功地用于体外受精卵的发育。在奥比希罗大学农场接受了三头小母牛的胚胎移植，其中一头怀孕了，并一直期待着3月23日的产犊。

In the first year (1987), the fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were invesigated. Cocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro and in vivo (rabbit oviducts) culture of in vitro fertilized and xenogenously fertilized oocytes, respectively. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%). However, the proportions of in vivo fertilized eggs (49.3%) and of cleaved eggs (34.6%) in the rabbit oviduct were higher (p<0.025) in the intra-follicular oocytes than those of the extra-follicular oocytes. These results demonstrated that the intra-follicular method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and cleavage than the conventional culture of extra-follicular oocytes.In the second year (1988), a co-culture system for oocyte maturation was established using granulosa cells in media (TCM 199 + 10% estrous cow serum). In vitro fertilization rate was improved by the use of swim-up and heparin treatment in frozen-thawed bovine spermatozoa (50 -60%). Presently, I have obtained rates of in vitro maturation (60 -70%), fertilization (50 -60%), cleavage (50%) and development into blastocysts (10 -20%) . For development of in vitro fertilized eggs, bovine oviduct epithelial cells were sucssesfully employed in media. One of three heifers received thransfer of in votro developed blastocysts was pregnant, and has been expecting the calving in march 23th, 1989 at the Obihiro University Farm.