Effects of isolation and culture of turkey primary follicular oocytes on morphology and germinal vesicle integrity

A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the i mpact of isolation and culture procedures on PFO integrity is lacking. This study describes the isolation and culture of PFOs from mature turkeys and the effects of these procedures on PFO morphology and germinal vesicle (GV) integrity. To isolate PFOs, ovarian cortex was incubated in trypsin-EDTA a lone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs, ranging in size from less than 100 mu m in diameter to 1,000 mu m, were recovered from each ovary. The culture of PFOs less than 100 mu m in diameter for 4 h resulted in blebbing of the oolemma followed by extru sion of ooplasm. Primary follicular oocytes 100 to 250 mu m in diameter sur vived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PF Os with intact granulosa cell investments survived longer than those fully or partially denuded of granulosa cells with CH. Co-culture of PFOs (100 to 250 mu m in diameter) on a monolayer of granulosa cells derived from matur e, yellow-yolk follicles augmented PFO survival rates. The rate of GV break down was not influenced by the isolation or culture of the PFO. These data provide the basis for developing procedures for the in vitro maturation and in vitro fertilization of isolated PFOs. Published by Elsevier Science Inc .