Fig. 2

Embryo development from ICSI to blastocyst stage. a ICSI was performed on MII oocytes with a Piezo-driven needle, the first polar is visible at 6 o’clock, a sperm is visible inside the ICSI pipette ready for injection. A two-cell embryo 48 h after ICSI (b). Cleaved embryos developed to the eight cell stage on average 5–6 days after injection (c) and were at pre-compaction stage on day 5–7 (d). Compacted morulae were visible on average at day 7–9 (e). The first blastocyst produced was a pure Southern White Embryo (fSWR5 × mSWR1, day 11 after ICSI) (f). The zona was removed by pronase digestion and embryo was left to expand for 2 days. From the isolated ICM of this embryo (g) and its twin obtained from the same group of oocytes (fSWR5 × mSWR1) two ES cell lines were derived. Subsequently Hybrid Southern White × Northern White blastocysts were obtained (h) (fSWR5 × mNWR1, day 9 after ICSI). One of these hybrid blastocysts (fSWR5 × mNWR2) naturally hatched from the zona at day 10 (i) and was used for ES cell derivation. One Southern blastocyst (fSWR4 × mSWR1) and two hybrid blastocysts (fSWR5 × mNWR2 and fSWR8 × mNWR2 (j)) were frozen with a standard slow freezing protocol after equilibration in 5% and subsequently in 10% of glycerol freezing media (fSWR8 × mNWR2 (k, l)). Scale bar corresponds to 100 µm