Non-Invasive Assessment of Spent Bovine Culture Media Using Proton Nuclear Magnetic Resonance.

Abstract

Early embryonic mortality is a great determinant of reproductive efficiency in mammals, including cattle. In in vitro production, approximately 90% of oocytes undergo fertilization following in vitro maturation; however, of these fertilized oocytes approximately 30-40% will reach the blastocyst stage. Factors involved in early embryonic mortality include oocyte and sperm quality, genetic factors, environmental and oxidative stresses, and largely in in vitro production, sub-optimal culture conditions. Current methods of embryo viability evaluation depend on morphological assessment, which in spite of its importance, is a poor predictor of embryo viability. Development of refined biological techniques like metabolomics has enabled us to explore the health of a cell based on its secreted metabolite constituents. Advanced analytical techniques, such as proton nuclear magnetic resonance (H1 NMR) has little chemical bias, gives detailed structural information of isolated metabolites and allow metabolites to be measured simultaneously with minimal sample preparation. We hypothesize that, embryos developing at different rates differ in their metabolomic signatures. The specific objective of study was to determine the metabolomic signatures of slow growing and fast growing embryos at timed stages of development, thereby determining possible biomarkers of embryo competency. Oocytes were collected from ovaries obtained from a local abattoir and then matured and fertilized in vitro using standard protocols. Presumptive zygotes were either placed for individual culture in 25 μl media drops. Media samples from fast growing embryos were collected at 2-cell (31 hours post fertilization), 4-cell (42 hours post fertilization), 8-cell (49 hours post fertilization) or 16-cell (72 hours post fertilization) and from slow growing embryos, which required additional 8 to 12 hours to reach equivalent embryo stage. The 18 μl media collection was diluted in 600 μl of 100% deuterium oxide (D2O) which included the internal reference standard TSP (sodium 3-(trimethyl-2,2,3,3)-1-propionic acid-d4). H1 NMR analysis was performed using a 600MHz Bruker NMR spectrometer. Preliminary data indicates distinct differences between metabolomic fingerprints of plain media, 2-cell, 4-cell, 8-cell, and 16-cell slow growing and fast growing groups. Specifically, slow growing embryos had lower lactate uptake and increased pyruvate production, while fast growing embryos demonstrated higher levels of pyruvate uptake. Moreover, in the presence of hydrogen peroxide, a reactive oxygen species, pyruvate can be decarboxylated to produce acetate, a metabolite which is higher in slow growing embryos compared to fast growing embryos. The data suggests that slow growing embryos have a slower metabolism and lower utilization of media substrates, as well as an increased response to oxidative stress. The results provide evidence towards the use of metabolomics for the development of a non-invasive tool for assessing embryo viability and the discovery of potential embryo viability biomarkers. (poster)