91 EMBRYO SUGAR CONSUMPTION DURING IN VITRO DEVELOPMENT

Abstract

Despite the presence of glycogen and triglycerides as energy reserves in bovine embryos, the viability of embryonic cells in culture is maintained primarily by energetic substrates present in the culture medium. The aim of this experiment was evaluate the embryo metabolism during three important stages of development: Days 1 to 3, Days 3 to 5, and Days 5 to 7; before the formation of Barr body, before the differentiation into ICM and trophectoderm, and during the blastocyst formation, respectively. In this study, we evaluated some energetic substrates (Krebs cycle intermediates). For these analyses we chose to use proton magnetic resonance (H¹-NMR) spectroscopy. Matured oocytes were purchased from DeSoto Biosciences (Seymour, TN, USA). The matured cumulus-oocyte complexes were fertilized in vitro according to our standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). For each group (Days 1–3, 3–5, and 5–7), the zygotes were place in individual drops, and after 48 h of culture, they were placed into well of the well (WOW) culture. For the 3–5 and 5–7 groups, the zygotes were first place in 400 μL of SOF and then moved in a single drop. The 5–7 group was not placed in the WOW system. Zygotes were incubated in a humidified mixture of 5% CO₂, 6% O₂, and 88% N₂ in air at 39°C. The embryos were scored for quality on the basis of morphological criteria. Samples of media (40 μL) were added to 560 μL of a stock solution prepared by dissolving 5.0 mg of sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate (TSP) in 50 mL of deuterium oxide. The TSP acted both as a chemical shift reference and as an internal standard for the purposes of quantitation. The resulting diluted samples were transferred to a 5-mm NMR tube. Samples were analysed on a Varian VNS-750 NB (750 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA). A total of 30 embryos were evaluated, 10 per stage. Data were statistically analysed through an analysis of variance using the Generalized Linear Model (GLM) procedure (SPSS, version 18, 2009). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α level was set at 0.05. All data were expressed as quadratic mean and mean standard error. The results, reported in Table 1, show that there was no statistical difference when we compare the lactate, myo-inositol, and pyruvate concentrations. However, there were significant differences when we focused on acetate and formate levels: after Day 3 the production of these two substrates significantly increased. Formate and acetate are important metabolites of the Krebs cycle. The changes of these two substrates are important signs that embryos start glycolysis after Day 3.

Table 1.  Quadratic means and mean standard errors of formate, lactate, myo-inositol, pyruvate, and acetate for Day 1–3, 3–5, 5–7, and control groups