Method for Isolating Immature Chicken Oocytes
Type of Degreethesis
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Knowledge of the development of the avian oocyte has been difficult to obtain due to the physical package of the oocyte at ovulation. The ability to isolate and culture avian oocytes, prior to the accumulation of large amounts of yolk and packing in albumen and shell, would allow the study of oocyte development. The objective of the described studies was to develop methods for dispersing and isolating the oocytes of immature chickens. Three concentrations of proteolytic enzymes were tested for efficiency for disaggregating ovarian tissue. Three methods were testing for removing contaminating erythrocytes (RBCs) and fibroblasts from the oocyte preparations. These isolation methods included: lysing of red blood cells and attachment of fibroblasts to a culture surface, Percoll density gradient centrifugation, and depletion of contaminating fibroblasts by binding to specific anti-fibroblast antibodies and lysing red blood cells. Increasing the concentration of Collagenase Type II in the enzyme mixture led to the release of a larger number of oocytes. While the lysing of red blood cells removed the red blood cells very effectively, depleting fibroblasts through attachment to the cell culture surface to allow decantation of the oocytes was not an effective method of disposing of the fibroblasts. Percoll density gradient centrifugation was effective in removing red blood cells from the oocytes. However, the remaining oocytes appeared to be damaged, and non-lysed fibroblasts remained in the solution. Antibody binding was a very effective method of removing fibroblasts from the cell solution, and the lysing of red blood cells in addition proved to be ideal for eliminating contaminating cell types. This experiment concluded that it is feasible to disperse the ovarian tissue in immature chicks and then isolate their oocytes for study. To analyze the immature oocytes further, flow cytometry was utilized to measure the DNA content of the cells. This enabled the assessment of the oocytes’ current position in the cell cycle. It was found that oocytes in the three-week-old chick have not yet undergone meiosis and are diploid. Studies focused on the cell cycle positioning of the immature oocyte could lead to significant advancements in reproductive efficiencies, and control of the offspring.