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

在第一年（1987年），研究了通过卵泡内或卵泡外方法成熟的牛卵母细胞的受精和分裂。将体外受精的卵母细胞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。与卵泡外法相比，卵泡内法没有提高核成熟率（57.9%和52.7%）。而卵泡内卵母细胞体内受精卵比例（49.3%）和卵泡外卵母细胞体内卵裂卵比例（34.6%）高于卵泡外卵母细胞（p<0.025）。这些结果表明，与传统的卵泡外培养相比，卵泡内培养的牛卵母细胞为细胞质成熟提供了一个生理环境，从而导致更高的受精率和卵裂率。第二年（1988年），以颗粒细胞为培养基（TCM 199 + 10%发情牛血清），建立了卵母细胞成熟共培养体系。在冷冻解冻的牛精子中使用游泳和肝素处理可提高体外受精率（50 -60%）。目前，我已经获得了体外成熟率（60 -70%），受精率（50 -60%），卵裂率（50%）和囊胚发育率（10 -20%）。牛输卵管上皮细胞在体外受精卵的培养中获得成功。1989年3月23日，在Obihiro大学农场，接受体外培养囊胚移植的三头母牛中有一头怀孕了，并期待着产犊。

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.