The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ
M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.
Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system
