Date of Award:

5-2024

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Irina Polejaeva

Committee

Irina Polejaeva

Committee

S. Clay Isom

Committee

Lee Rickords

Committee

Ralph Meyer

Committee

Gregory Podgorski

Abstract

Assisted reproductive technologies (ART) have become a valuable tool in the production of animals and also treatment for human infertility. In animal production specifically, somatic cell nuclear transfer (SCNT), or cloning, is important for the production of transgenic or high genetic value animals. Oocyte maturation is a necessary and critical step in the production of embryos and greatly impacts the success of all subsequent ART techniques. While in vitro maturation is a routinely used technique, improvements are needed to enhance the efficiency in producing embryos. There is also a need to develop non-invasive methods to assess the quality of oocytes and embryos and determine their chances to produce a live animal or child. The work within this dissertation is aimed at addressing both of these issues and may be applied to improve animal production and also human infertility.

Many livestock oocytes can be collected from abattoir derived ovaries but must be matured in the lab. Many techniques have been used to improve the number and quality of mature oocytes. Recently, the cytokine (FGF2, LIF and IGF1) supplemented medium termed ”FLI” medium has shown promise using pig oocytes. We aimed to test the effects of these cytokines on cattle oocyte maturation due to the importance cattle have in agriculture. We found that FLI medium improved the quantity and quality of mature oocytes and embryos as well as effected the expression of developmentally important genes. When these embryos were transferred to recipients it also resulted in a significant increase in live animals born.

We also investigated the use of Raman spectroscopy as a non-invasive method to determine the developmental potential of cattle embryos. Raman spectroscopy measures vibration of chemical bonds and can be used to measure metabolites within embryo culture medium. Spent culture medium is a good source of information on embryos and is usually discarded after embryo culture. We found that Raman spectroscopy, along with machine learning, can determine culture medium that contained embryos that developed successfully to the blastocyst stage compared with those that did not.

Cumulus cells, which surround and support the oocyte, are also a good source of information that can be used as a non-invasive method of developmental potential. Cumulus cells also play an important role in oocyte maturation, with signals going from cumulus cells to the oocyte and from oocyte to cumulus cells. We used single-cell RNA sequencing to look at differential gene expression of cumulus cells from oocytes matured in FLI medium compared to control medium. In cumulus cells from the FLI group, we saw differential gene expression in several important cellular pathways and also expression of genes associated with improved developmental potential.

Overall, this work has increased our efficiency in producing cloned cattle. We also have observed differential gene expression in oocytes, embryos and cumulus cells that may be important in oocyte maturation and embryo development.

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