Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

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Abstract
Design by supplementing each with 15 mg bovine serum albumin (BSA)/ml or 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 \g=m\ginsulin/ml, 5 \g=m\gtransferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epi- dermal growth factor (EGF)/ml. One-cell embryos were cultured at 39\s=deg\Cwith 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P < 0\m=.\001)when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P > 0\m=.\37)from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein\x=req-\ free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo develop- ment from the 1-cell to the hatching blastocyst stage in a defined protein-free culture

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