Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Joseph D. Roussel

Abstract

A reliable procedure was developed to harvest purified populations of bovine uterine epithelial cells from the luminal endometrium that allowed cells to be maintained in vitro for multiple subpassages. This method allowed adequate numbers of cells for in vitro culture studies to evaluate uterine secretion rates, uterine-embryo interactions and a new embryo co-culture system. In a second study, cells were isolated from the uterus and oviducts collected from cattle on day of estrus, on d 4 to 6, d 8 to 10 and d 14 to 16 of the cycle to monitor growth and development following incubation in vitro. The highest percent cell viability and attachment during primary culture was noted for cells isolated on the day of estrus or between d 4 to 6 for both uterine and oviductal cell populations. The effects of different culture media and incubation temperatures on growth and proliferation of uterine and oviduct epithelial cells in vitro were evaluated. Cells incubated in CMRL and MB2 had the highest growth rates, however, there were no differences in proliferation rates among media evaluated. There were no differences in growth and proliferation rates between different incubation temperatures (37$\sp\circ$C vs 39$\sp\circ$C). Morphological observations of cell integrity and quality were made using image analysis during primary culture and following the first and third subpassages. Optical density readings were influenced by cell subpassage and quality. Cell quality following in vitro culture could be effectively monitored using image analysis. Bovine oocytes were matured and fertilized in vitro in different culture media. Culture medium did not influence fertilization or cleavage rates. Early embryo development was enhanced following incubation in CMRL and TCM media. Embryo viability percent improved when morulae were maintained on oviductal cells compared with those cultured in medium alone. Bovine embryos derived from in vitro-fertilization procedures were co-cultured with oviductal cells isolated between cycle d 4 to 6 or 14 to 16 and proteins secreted by oviductal cells monitored. Cycle stage oviductal cells were isolated did not influence developmental rates or proteins secreted. However, co-culture with oviduct epithelial cells were superior to culture in medium alone.

Pages

146

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