|Potts, W - SAS LABORATORY|
Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 15, 1998
Publication Date: N/A
Interpretive Summary: The transfer of genes expressing traits of economic interest is extremely difficult in birds due to the size of the hen's ovum and inaccessibility to the ovum's nucleus. A novel approach to the production of transgenic poultry is to use microscopic immature oocytes isolated from the ovary. These oocytes theoretically could be matured to where hey can be subjected to in vitro fertilization procedures. Using micromanipulation technology developed for mammalian oocytes, foreign genes can then be transferred into these hen oocytes. This study describes the isolation and culture of immature ovarian oocytes from mature turkeys and the effects of these procedures on oocyte viability. Using enzymatic digestion, about 200 to 500 oocytes, ranging in size from less than 100 um in diameter to up to 1,000 um, were recovered from each ovary. Isolated oocytes larger than 350 um survived better in culture than smaller oocytes. These observations provide to other scientists basic information for the development of procedures for the maturation and in vitro fertilization of avian oocytes, which may ultimately lead to new approaches to the production of transgenic poultry.
Technical Abstract: A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the impact of isolation and culture procedures on PFO integrity is absent. 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 alone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs ranging in size from less than 100 um in diameter to up to 1,000 um, were recovered from each ovary. The culture of PFOs less than 100 um in diameter for 4 h resulted in blebbing of the oolemma followed by extrusion of ooplasm. PFOs 100 to 250 um in diameter survived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PFOs 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 um in diameter) on a monolayer of granulosa cells derived from mature, yellow-yolk follicles augmented PFO survival rates. The rate of GV breakdown 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.