Date of Award:

12-2025

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Irina Polejaeva

Committee

Irina Polejaeva

Committee

Ying Liu

Committee

Lee Rickords

Abstract

In vitro embryo production (IVEP) is a groundbreaking assisted reproductive technology that has transformed the livestock industry and improved reproductive efficiency. IVEP involves collecting eggs and growing them in a laboratory until they are mature enough to be fertilized and develop into embryos. However, the efficiency of these procedures in sheep IVEP remains low compared to other livestock species. The goal of this study was to identify and optimize sperm preparation and fertilization conditions for more predictable development of sheep embryos, so that the effect of sperm from different genetic backgrounds on embryo development, embryo quality, developmental timing, and survivability and re-growth patterns after freezing could be explored. Sperm motility was assessed across four pH levels (a standard non-buffered control and three buffered media) and two laboratory culture methods. The optimal fertilization method was determined by comparing the developmental rates and embryo quality of embryos from two non-gene-edited and two gene-edited rams with the sickle-cell disease (SCD) mutation. The most favorable fertilization method was then used to evaluate the effects of sperm genotype on developmental timing and survival after freezing. Buffered media at physiological pH (7.00 and 7.40) resulted in the highest percentage of progressive sperm motility. The microdrop fertilization method showed a trend toward increasing sperm survivability over time, but the culture system did not affect overall embryo development or quality. Gene-edited sperm had produced more late-stage embryos in the microdrop system compared to the non-gene-edited ram. Heterozygous SCD embryos from the Romney breed had a lower ratio of fetal to placental precursor cells compared to embryos from a non-gene-edited ram of the Polypay breed. However, embryo quality did not differ between non-gene-edited and sickle-cell disease gene-edited embryos within the same breed. Sperm genotype did not influence developmental timing or post-freezing survival and re-growth patterns. Overall, the findings in this study provide insights that serve as a foundation for further refining sheep in vitro fertilization protocols and may be implemented in the agricultural industry and in laboratory breeding to increase production of gene-edited animals for biomedical research that benefits society.

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