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

第一年（1987年）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。卵母细胞在体外或在兔输卵管中受精，并分别在体外和体内（兔输卵管）培养体外受精和异种受精的卵母细胞后评估卵裂。与卵泡外培养法相比，卵泡内培养法并没有提高核成熟率（57.9%和52.7%）。但卵泡内卵母细胞的体内受精卵比例（49.3%）和输卵管内卵裂卵比例（34.6%）均显著高于卵泡外卵母细胞（P<0.025）。第二年（1988年）建立了以颗粒细胞为培养液（TCM 199 + 10%发情期牛血清）的牛卵母细胞共培养体系。采用上游法和肝素处理，可提高牛精子冻融后的体外受精率（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发育中获得成功。1989年3月23日，在带广大学农场，3头接受体外发育囊胚移植的小母牛中有1头妊娠，并期待产犊。

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.