Masters Theses

Date of Award

12-2023

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

Daniel J. Mathew

Committee Members

J. Lannett Edwards, Agricola Odoi, Lew Strickland

Abstract

Histotroph, secreted from bovine endometrial epithelial and stromal fibroblast (SF) cells, supports embryo development prior to implantation. Additionally, secretions from bovine oviduct epithelial cells can help prime early embryos for development after entering the uterus. We hypothesized that culturing in vitro produced (IVP) bovine embryos in potassium simplex optimized medium (KSOM) conditioned with bovine oviduct and endometrial cells will increase IVP embryo development rate, timing of embryo structure formation, embryo cell numbers and quality compared to non-conditioned KSOM. Primary oviduct epithelial (OEp) cells and endometrial epithelial (EEp) and stroma fibroblast cells (SF) were isolated from six female bovine reproductive tracts. Potassium simplex optimized medium was then cultured with the cells or without the cells for 12 h to produce cell conditioned and non-conditioned KSOM (CON), respectively. To begin the study, in vitro produced bovine embryos were developed using standard in vitro production methods and cultured in CON or OEp conditioned KSOM from Days 1 to 3 (n=1356) when they were manually checked for cleavage. At which point, the CON treated embryos were then subdivided and cultured again in CON or in EEp conditioned KSOM or conditioned KSOM created by EEp and SF combination culture (EEp/F) from Days 4 to 8 (n=385) in an embryo time lapse monitoring system, the MIRI TL6. To evaluate the combined effect of culturing IVP embryos sequentially in OEp conditioned KSOM followed by endometrial cell conditioned KSOM, embryos cultured in the OEp conditioned KSOM from Days 1 to 3 were then cultured in EEp or EEp/F conditioned KSOM from Days 4 to 8 (n=77) in the MIRI TL6. In the MIRI, embryo development was monitored in real time between Days 4 and 8. On Day 8, embryo diameter and quality was scored and all blastocysts were removed from the MIRI for inner cell mass (ICM) and trophectoderm (TE) cell counts using immunohistochemistry. There was no effect of OEp on cleavage and 8-16 cell embryos on Day 3, but embryos cultured in OEp conditioned KSOM did have improved compact morula and early blastocyst rate and time to structure formation between Days 4 to 8 compared to embryos culture in non-conditioned KSOM (P < 0.01). Normal and expanded blastocyst rates between Days 4 and 8 were similar for embryos cultured in OEp and CON between Days 1 to 3, however, embryos cultured in OEp from Days 1 to 3 had greater normal and expanded blastocyst rates when compared to embryos cultured completely in CON from Days 1 to 8 (P < 0.01). Compared to embryos cultured in CON from Days 1 to 8, embryos cultured in EEp or EEp/F conditioned KSOM from Days 4 to 8 had greater compact morula and early blastocyst rates and time to morula and blastocyst formation as well as normal and expanded blastocyst rates (P < 0.01). Finally, embryos cultured in OEp from Days 1 to 3 followed by EEp or EEp/F (OEp+) from Days 4 to 8 had the greatest embryo development rates (compact morula through expanded blastocyst stages) and reduced time to structure formation (compact morula through early blastocyst stages) compared to embryos cultured in CON from Days 1 to 8 (P < 0.05). Conditioned media had no effect on embryo diameter or cell number; however, embryo quality score was numerically less (indicative of better quality) for embryos cultured in OEp from Days 1 to 3 followed by EEp from Days 4 to 8 compared to nearly all other treatments. Thus, sequential culture of IVP bovine embryos in oviduct followed by endometrial cell conditioned KSOM can improve IVP early embryo development in cattle compared to non-conditioned KSOM and can be used to improve IVP embryo numbers and possibly, survival.

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