Non-Invasive Analysis of Gamete Metabolites During In Vitro Embryo Production Using Nuclear Magnetic Resonance

Marcello Rubessa, Andrea Ambrosi, Scott E. Denmark , Matthew B. Wheeler

The in vitro production of bovine embryos has dramatically increased in recent years. From the first calves produced entirely from IVEP (in vitro embryo production)until today, the in vitro embryo production systems have been modified and improved. The media for embryo production generally are formulated with nutrient excess and furthermore it is known that excess nutrients can have negative effects on embryo development. In order to assess the actual metabolic needs of the gametes and embryo, we decided to evaluate energy consumption with a non-invasive technique, proton nuclear magnetic resonance (1H-NMR) during gamete co-incubation. In the first experiment we evaluated the behavior of cumulus oocytes complex (COCs), sperm and COCs plus sperm. We chose to evaluate pyruvate, lactate and alanine. The effect of the different groups was significant only for pyruvate (p>0.05) otherwise we did not find any difference in alanine and lactate concentrations. The concentration of pyruvate is significantly decreased in COCs and semen + COCs. After this experiment we re-formulated the IVF (in vitro fertilization) medium by changing the concentration of pyruvate and lactate (to 1/3 and 1/10 of standard concentrations) based on the metabolic results. The reduction of pyruvate and lactate had a positive effect on embryo production, which was evident by the increase (P<0.05) of advanced stage embryos in the modified vs. control media (39.6% vs. 30.4% respectively). These results suggest that this new formulation improves embryo development. These results can be used to change recommended concentrations of these substrates in IVF media.