34 PRE-IMPLANTATION DEVELOPMENT OF HORSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS ORIGINATED FROM METAPHASE I OOCYTES

Abstract

Because of the limited availability of horse oocytes and their wide maturation window that leads to the existence of a subpopulation of oocytes with significantly longer maturation period, horse cloning would benefit from the possibility of also using metaphase I oocytes (Choi et al. 2009 Cloning Stem Cells). The scope of this work was to compare the developmental ability of cloned horse embryos constructed using oocytes in metaphase I (MI) and II (MII). Oocytes of slaughtered mares were matured for 27 h in DMEM/F12 with 10% FCS, 1 µL mL^–1 of insulin-transferrin-selenium (ITS), 1 mM sodium pyruvate, 50 ng mL^–1 of long-epidermal growth factor, 100 ng mL^–1 of long-insulin-like growth factor-I, and 0.1 IU mL^–1 each of FSH and LH, denuded of cumulus and enucleated using a zona-free method (Lagutina et al. 2005 Reproduction). Oocytes with a visible polar body were classified as MII and those with no polar body as potential MI. During enucleation, oocytes having a metaphase plate only were confirmed as MI; oocytes in anaphase and telophase were classified as MII. Adult skin fibroblasts of passages 2 to 10 were cultured in TCM 199/DMEM with 10% FCS and serum starved for 1 to 2 days before NT. Nuclear-transfer embryos were constructed after washing of zona-free oocytes in 400 µg mL^–1 of phytohemagglutinin P and attachment of each to a single cell in HEPES-SOF by fusion with 2 direct-current (DC) pulses of 1.2 kV cm^–1 applied for 30 µs in fusion medium. One hour after fusion, embryos were activated by 5 µM ionomycin for 4 min, followed by culture in 5 µg mL^–1 of cycloheximide and 1 mM DMAP in SOFaa for 3 h. Embryos were cultured in SOFaa in 5% CO₂ and 5% O₂ at 38.5°C. Half of the medium was renewed on Day 3 and replaced on Day 5 with DMEM/F12 with 5% FCS and 5% serum replacement. Cleavage was assessed 48 h after activation and the rate of blastocyst formation was recorded at Day 8. The data were compared by chi-square test. The development ability of MI embryos assessed by cleavage and blastocyst formation was significantly lower than of MII embryos (Table 1). The obtained MI blastocysts were smaller than their MII counterparts. These data demonstrate that MI oocytes account for 20% of the total oocytes after 27 h of maturation, have a lower developmental competence to form blastocysts after NT, and the blastocysts obtained are of smaller size and likely less viable. Therefore, the use of MI oocytes can only marginally improve the outcome of horse cloning.

Table 1.  Embryo development after SCNT using oocytes in metaphase I or II