Background

Embryo and Sperm Cryopreservation Background

Background

The exponential growth of mutant mouse lines and the number of animals required for definitive studies has led to exponential growth in animal costs and challenges for colony management. Cryopreservation of mouse germplasm (sperm and embryos) is an efficient and cost-effective solution for managing colony size. In addition, this service permits archiving of transgenic models not currently needed but potential invaluable to future studies and provides protection against spontaneous mutations occurring in subsequent generations maintained in the live colony. Each method of cryopreservation of sperm or embryos has advantages and disadvantages and can be used depending on your lab’s requirements.

Sperm cryopreservation:

Sperm cryopreservation is more cost-effective than embryo cryopreservation. The sperm from a single male mouse could potentially give rise to as many as 20 times more offspring than embryos from a single female donor (Sharp and Mobraaten, 1996; Sztein et al., 1997). However, reconstituting live mice from frozen sperm requires in vitro fertilization (IVF) and it is more expensive compared with reconstitution via frozen-thawed embryo transfer (Critser, Mobraaten, 2000). Furthermore, reconstituting live mice by IVF is inefficient with rare strains.  Currently, almost all cryopreserved mutant mouse lines at OHSU are preserved through sperm cryopreservation.

Embryo cryopreservation:

Cryopreservation of mouse strains has traditionally relied on freezing embryos at the 2-cell or 8-cell stage, mainly because live mice can easily be reconstituted by embryo transfer (Whittingham et al., 1972). This method is highly beneficial when the mouse lineage exhibits two or three distinct mutations, especially in cases where one or more mutation(s) are homozygous.

IVF embryo cryopreservation (Speed Cryo):

The freshly collected sperm from 2-3 transgenic males is used to fertilize the eggs of superovulated females of the same strain background via in vitro fertilization and then cryopreservation of 2 cell or 6-8 cell stage embryos is performed. Simultaneously we also freeze 10 samples of sperm from these males. Thus, your strain will be cryopreserved and stored as both embryos as well as sperm.

Cryo-preservation accessories used in our laboratory. (A) Eight cell embryos prepared for cryopreservation. (B) Plasticaccessories listing from top to bottom: concave cryogenic vials used for freezing ES cells, a conical cryogenic vial used for freezing of embryos/sperm/oocytes, cryogenic straws used for freezing of embryos/sperm/oocytes. (C) FTS controlled Freezer for embryo cryopreservation. (D, E) Taylor-Wharton extended time liquid nitrogen refrigerator and cryostorage system used for long term storage of germplasm.

Development of mouse embryo after in vitro fertilization. From left to right: IVF process, 2-cell stage embryo, 8- cell morula, and blastocyst.