EFFECT OF VARIOUS GROWTH FACTORS IN A DEFINED CULTURE MEDIUM ON IN VITRO DEVELOPMENT OF BOVINE EMBRYOS MATURED AND FERTILIZED IN VITRO

摘要

Bovine embryos matured and fertilized in vitro were cultured in a simple, protein-free, chemically defined medium consisting of synthetic oviduct fluid medium (SOFM) modified by substituting polyvinyl alcohol (PVA) for BSA and supplementing with Basal Medium Eagle (BME) amino acids, and glutamine, to investigate the effects of standard fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor pi (TGF beta 1) on bovine embryonic development. In Experiment Ia, when the 2-cell, 8-cell, morula and early blastocyst stage embryos obtained from the culture in human serum (HS)-supplemented SOFM were cultured singly in growth factor-supplemented medium (SOFM+PVA) for 48 h and further cultured in SOFM+PVA without growth factor, FGF and EGF significantly (p<0.05) increased development to blastocysts of morula (73.3%) and early blastocyst embryos (84.4%) compared to control (51.1% and 60.0% for morula and early blastocyst, respectively), and SOF+HS (positive control) showed significantly higher developmental rates than control in all stages of embryos (p<0.05). However, supplementation with IGF-I or TGF beta 1 did not improve embryo development in all stages of embryos compared to controls. In Experiments Ib, II and III, 2-cell embryos were selected after 30 h of IVF and then cultured in a defined medium to investigate the effects of single or group culture, different doses of FGF and the synergistic effect of EGF and FGF on embryo development. There was no significant difference in developmental rate to blastocysts with the number (single or group) of embryos cultured per well and the doses (0, 0.05, 1, and 10 ng/ml) of FGF. The medium supplemented with both FGF and EGF showed significantly (p<0.05) higher developmental rate to blastocysts (31.4%) of 2-cell embryos cultured in a group than control medium (22.0%), and FGF and/or EGF increased the cell number of blastocysts(93.6+/-7.0. 90.5+/-4.7 and 87.3+/-3.8 for EGF, FGF, and EGF and FGF, respectively) compared to controls(73.5+/-3.1). The results indicate that the supplementation of FGF, EGF of both in a defined culture medium can improve the in vitro development of bovine embryos to morula and blastocyst stages; FGF and EGF may act synergistically on bovine embryo development in vitro.