Development in vitro of investment‐free marsupial embryos during cleavage and early blastocyst formation

Twenty embryos at the cleavage-arrested four-cell stage of the brown antechinus, Antechinus stuartii, were used to develop a method to obtain investment-free embryos by incubation at 35 degrees C in 2.5% pancreatin, 0.05% trypsin, and 0.05% pronase. Control intact embryos and the investment-free embryos were incubated in Dulbecco's Modified Eagle's Medium with high glucose and 10% foetal calf serum in 5% CO2 in air at 35 degrees C for 48 h. Release of embryos from the investments (shell, mucoid, and zona pellucida) took 4-4 1/2 h in pancreatin, 6 h in trypsin, and 55 min to 1 1/4 h in pronase. Embryonic survival in vitro was best following pronase digestion. Pronase concentrations of 0.05%, 0.1%, 0.25%, 0.5%, and 1.0% were used to free one- to 16-cell stage embryos of brown antechinus and the stripe-faced dunnart, Sminthopsis macroura, from their investments. Postincubation survival and development of embryos in vitro was best using between 0.05% and 0.25% pronase. Blastomeres in both intact and investment-free embryos shared similar characteristics during cleavage and blastocyst formation. The combined effect of these characteristics was that in intact embryos, no morula was formed, and the blastocyst developed, when cell numbers were high enough (about 32 cells), as a unilaminar structure flattened against the zona pellucida and without an inner cell mass. In investment-free embryos, the blastomeres dispersed over the base of the culture vessel. The investments were necessary to confine the blastomeres, to provide a surface for blastomere flattening, and to allow normal cellular associations to develop.