92 Altering carbohydrates during embryo culture affects quality and gene expression of the resulting blastocysts

The objective of this study was to determine the effect of different carbohydrate combinations and concentrations in culture media on embryo development and blastocyst sex. Cumulus–oocyte complexes (COCs) were obtained from a local abattoir, matured for 22 h, and fertilized using Holstein semen from a singular lot. Gametes were co-incubated for 18 h, then denuded and placed in culture medium 1 (IVC1). After 72 h, IVC1 was replaced with culture medium 2 (IVC2). Control IVC1 medium contained 0.5 mM glucose, and control IVC2 medium contained 3 mM fructose. For Experiment 1, IVC1 medium was compared with a modified medium containing 0.15 mM glucose + 0.28 mM myo-inositol. On Day 7, blastocysts (BLs) were collected for embryo sexing (control, n = 166; altered, n = 181) to determine the sex ratio of BLs. Embryos were washed three times in Dulbecco’s PBS with polyvinylpyrrolidone and lysed in lysis buffer at 65°C for 15 min and 98°C for 2 min. Then qPCR was performed using TaqMan and X-specific PLP and Y-specific SRY primers. In Experiment 2, control IVC1 medium was compared with three different IVC1 media: (1) 0.15 mM glucose + 0.3 mM maltose; (2) 0.15 mM glucose + 0.3 mM galactose; and (3) 0.15 mM glucose + 0.15 mM maltose + 0.15 mM galactose. Four embryos were individually collected per group, and qPCR was performed using a TaqMan assay for genes related to cell lineages, endoplasmic reticulum stress, and oxidative stress. For Experiment 3, control IVC2 medium was compared with media containing (1) 0.3 mM maltose, (2) 0.3 mM galactose, or (3) 0.15 mM maltose and 0.15 mM galactose, in addition to the existing 3 mM fructose in the IVC2 medium. The cell lineage and stress assay was performed as stated above. All experiments were repeated four times using 75–150 COCs per group. Data were analyzed via one-way ANOVA, using the PROC GLM procedure of SAS v 9.4, using the pdiff option of LSMEANS. For Experiment 1, the modified medium had no effect on embryo development (21% grade 1 and grade 2 [G1+G2] BLs for both groups). The percentage of female BLs was 59% for control and 61% for the modified medium, and the percentage of female embryos in each stage did not differ between groups. However, control medium produce more expanded BLs than the modified medium (39% vs. 25%, P = 0.02). In Experiment 2, control G1+G2 BLs were 23%, and groups 1–3 were 30%, 14%, and 18%, respectively. Group 1 yielded more G1+G2 BLs compared with all other groups (P = 0.004) and more G1 BLs than groups 2 and 3 (P = 0.02). Gene expression was similar between groups for all genes except SOD1, an oxidative stress gene, for which group 1 and group 3 had less expression than the control. In Experiment 3, control had 21% G1+G2 BLs, and groups 1–3 were 20%, 16%, and 22%, respectively; no statistical differences were found. In general, groups 1 and 3 had decreased expression of stress and cell differentiation markers (P < 0.05). In conclusion, the combination of glucose and myo-inositol did not affect BL sex ratios, and the combination of glucose and maltose in IVC2 medium increased G1+G2 BLs, while decreasing embryonic stress. Further characterization of the use of maltose or maltose+galactose in IVC2 medium should be performed regarding their effect on embryonic stress and cell differentiation.