Culture of Preimplantation Rabbit Embryos

It is surprising that so little attention is currently given to in vitro culture of preimplantation rabbit embryos, even though the rabbit is the only laboratory animal in which there is very considerable embryo growth before implantation, resulting in a 300-fold increase in protein content of embryonic cells during the preimplantation period and the formation of more than a 100,000 cells in the blastocyst. This growth pattern explains why blastocyst formation in vitro has an absolute requirement for amino acids, and vitamins, particularly inositol, are esssential for blastocyst growth. A semi-defined medium supplemented with 1.5% BSA (variously known as BSM II or modified F10) was developed at Cornell University at the end of the 1960s and allowed the systematic investigation of the requirements for development of 1-cell rabbit embryos to blastocysts. However, the requirements for in vitro blastocyst growth comparable to in vivo growth still remain an unsolved problem. Citrate, often found as a contaminant in serum albumin, may have an essential role in rabbit blastocyst growth, which would fit in with its role in the development of serum-free media for culture of various types of mammalian cells.A comprehensive account of the methodology is given to enable a researcher with experience culturing embryos of a different species to work on the rabbit embryo. This account covers medium preparation, hormonal stimulation of superovulation, natural breeding/artificial insemination, and collection of embryos of different stages from 1-cell to blastocyst either after euthanasia or under anesthesia. Peculiarities of the rabbit embryo such as the presence of the mucoprotein coat and its effects on behavior of cultured and transferred embryos are described. Suggestions are made for future avenues of research.