ENHANCING MEDIA COMPOSITION OF IN VITRO DERIVED BOVINE EMBRYOS

In vitro production (IVP) of bovine embryos has had a 12% annual growth rate over the past few decades. Over one million IVP embryos were produced worldwide in 2019 with North America remaining one of the leaders in this technology. However, embryo yield and quality and calf production utilizing IVP procedures is suboptimal. ~35% of in vitro derived embryos are expected to reach the blastocyst stage compared to > 70% of in vivo derived embryos. Only 25% of cattle receiving an IVP embryo will produce a live calf. Therefore, improving IVP of bovine embryos is crucial to improve economic efficiency for producers.I propose that a major reason why these insufficiencies exist in in vitro culture conditions is because they do not adequately mimic the in vivo environment. Moreover, in vitro embryos are exposed to atmospheric oxygen conditions, light, and alterations in temperature as they are manipulated during IVP. This can lead to the accumulation of reactive oxygen species (ROS). ROS are normal by-products from oxygen metabolism utilizing the electron transport chain and are normally regulated by factors within the reproductive tract. This leads to the accumulation and imbalance of free oxygen radicals and the subsequent inactivation of enzymes, peroxidation of the lipid membrane, DNA alteration, and apoptosis.I hypothesize that the inclusion of antioxidants in IVP cultures will improve embryo production and embryo quality. Defining important antioxidants to include in culture media is crucial to improve blastocyst development and subsequent calf production.The major goals of this project are:Assess embryo development and quality when culturing with antioxidants.Evaluate cleavage and embryo development at days 2, 5, 7, and 8 of culture.Evaluate cell proliferation and DNA fragmentation with optimized TUNEL and PCNA staining.Stain embryos with immunofluorescence to identify trophectoderm and inner cell mass cells.Snap-freeze embryos to sex and use for RNA-seq for transcriptome analysis.Assess embryo development and quality when culturing with altered media composition.Evaluate cleavage and embryo development at days 2, 5, 7, and 8 of culture.Evaluate cell proliferation and DNA fragmentation with optimized TUNEL and PCNA staining.Stain embryos with immunofluorescence to identify trophectoderm and inner cell mass cells.Snap-freeze embryos to sex and use for RNA-seq for transcriptome analysis.Analyze early pregnancy outcomes of embryos cultured with antioxidants and/or altered media composition.Freeze embryos using vitrification. Optimization and design of vitrification process is needed.Develop proficiency in embryo transfer.Develop proficiency in embryonic rectal palpation via ultrasounding.