DEVELOPMENT OF IN VITRO MATURATION TECHNOLOGY FOR GIBEL CARP OOCYTES

In vitro oocytes maturation is a key and efficient technology for gene and cellular micromanipulation in oocytes of fish. In this study, we used gibel carp oocytes as studying materials to develop an efficient in vitro induction maturation technology. Based on previously established germinal vesicle (GV) isolation and premeiotic chromosome preparation methods , four different stage oocytes including GV0, GV1, GV2 and GV3 were firstly distinguished by GV migration and premeiotic chromosome condensation status, and the maturation rate (%), cleavage rate (%) and hatching rate (%) were analyzed. GV1, GV2 and GV3 oocytes had the capacity for in vitro induction maturation, and GV1 oocytes were the optimal stage oocytes because they have longer time for micromanipulation. We found that the optimal induction conditions for GV1 oocytes in vitro were 12 hours at 23℃ in pH 8.5 Gey’s balanced salt solution(GBSS) with 1 μg/ml DHP (17α, 20β-dihydroxy-4-pregne-3-one), by which up to 55.5% hatching rate was reached from these stripped follicle membrane oocytes. Moreover, we injected the GFP-zfh2af1o mRNA into the GV1 oocytes, and found that the whole process of in vitro maturation, fertilization and embryo development of the micro-manipulated GV1 oocytes could be tracked by the expression of GFP, and normal fertilization and embryo development could be produced from the induced mature eggs. Therefore, we established an efficient platform for studying oocyte development and gene and cellular micromanipulation in this fish.